Methods for the search of quinoproteins

STELLINGEN

1

Gezien d e r e s u l t a t e n g e p r e s e n t e e r d d o o r T ü r en Lerch m e t b e t r e k k i n g t o t d e P Q Q d e t e c t i e in b e n z y l a m i n e o x i d a s e uit Pichia pastoris w o r d t e e n s t e m e e r duidelijk h o e b e l a n g r i j k een b l a n c o e x p e r i m e n t kan zijn.

- S.S. TUr en K. Lerch (1988) FEBS L e t t . 238, 7 4 - 7 6 .

2

In h e t l i c h t van d e m o g e l i j k e r o l van s e r u m a l b u m i n e a l s a - s p e c i f i e k e t r a n s p o r ­ t e u r voor P Q Q lijkt d e k e u z e van d i t eiwit a l s v o o r b e e l d t e r i l l u s t r a t i e van de o n g e s c h i k t h e i d van f l u o r e s c e n t i e s p e c t r o s c o p i e voor P Q Q d e t e c t i e , n o g a l o n g e l u k -kig.

- O. Adachi, E. S h i n a g a w a , K. M a t s u s h i t a , K. N a k a s h i m a en M. A m e y a m a (1989) P r o c e e d i n g s of t h e f i r s t i n t e r n a t i o n a l s y m p o s i u m on P Q Q a n d q u i n o -p r o t e i n s (J.A. J o n g e j a n a n d J.A. Duine, e d s . ) . K l u w e r A c a d e m i e P r e s s , D o r ­ d r e c h t , p g . 145-147.

M.A.G. van Kleef, P. D o k t e r . A.C. M u l d e r en J.A. Duine (1987) A n a l . B i o -c h e m . 162, 143-149.

3

Dat d e t o e v o e g i n g van b e p a a l d e a m i n o z u r e n aan b a t c h - o f k o o l s t o f - g e l i m i t e e r d e k o n t i n u e c u l t u r e s van Hyphomicrobium X geen t o e n a m e van h e t d r o o g g e w i c h t t e n ­ g e v o l g e heeft, m o e t w o r d e n t o e g e s c h r e v e n aan h e t o n v e r m o g e n van d e z e b a c t e r i e om d e b e t r e f f e n d e a m i n o z u r e n in zijn eiwit in t e b o u w e n .

- J.B.M. Meiberg (1979) PhD T h e s i s , University o f G r o n i n g e n . - M.A.G. van Kleef en J.A. Duine (1988) FEBS L e t t . 237, 91-97.

4

Voor G r a m - n e g a t i e v e b a c t e r i ë n is d e g r o o t t e van e n e r z i j d s d e a f s t a n d t u s s e n de b u i t e n m e m b r a a n en d e c y t o p l a s m a m e m b r a a n (7 nm) en van a n d e r z i j d s h e t v o l u m e van h e t p e r i p l a s m a (20 % van h e t c e l v o l u m e ) niet m e t e l k a a r in o v e r e e n s t e m m i n g .

- J . B . S t o c k , B. Rauch en S. R o s e m a n (1977) J. Biol. C h e m . 252, 7 8 5 0 - 7 8 6 1 . - D.B. Oliver (1987) in: Escherichia coli a n d Salmonella typhimurium, c e l l u l a r

a n d m o l e c u l a r b i o l o g y (F.C. N e i d h a r d t e d . ) . Vol. 1. A m e r i c a n S o c i e t y f o r M i c r o b i o l o g y , W a s h i n g t o n DC, p g . 56-69.

- S.J. F e r g u s o n (1988) in: Bacterial e n e r g y t r a n s d u c t i o n (C. A n t h o n y ed.). A c a d e m i e P r e s s , L o n d o n , p g . 151-182.

s

Bij het ontwikkelen van een h.p.l.c. analyse methode wordt in het algemeen te

weinig aandacht geschonken aan de verhelderende rol die t.l.c. hierbij kan spelen.

6

De steeds verdergaande verzuring van ons milieu kan bij de mens uiteindelijk lei­

den t o t ernstige aandoeningen van het centrale zenuwstelsel zoals dementie en

geheugenverlies.

7

Alvorens conclusies te verbinden aan de karakteristieken van het residue na simu­

latie van absorptiespectra met "lognormaal curven", verdient het aanbeveling eerst

deze bewerking met bekende spectra uit te voeren.

- D.E. Metzier, C.M. Metzier en J. Mitra (1986) Trends Biochem. Sc. //,

157-159.

8

In tegenstelling tot de meer serieuze literatuur heeft de afbeelding op de omslag

van goedkope detective romans niets met de inhoud te maken.

9

De door de overheid opgelegde dwang voor basisscholen om bij aanschaf van een

computer te kiezen voor een IBM-achtige voorkomt weliswaar een versnippering

van kennis en investeringen, maar is uit onderwijskundig oogpunt een slechte

keuze.

10

Het in speelgoedwinkels en modelspoorzaken verkrijgbare "scener> materiaal" is

voornamelijk bedoeld om de modelspoorbouw belachelijk te maken.

11

De analyseresultaten van het Groningse drinkwater van medio april 1989 laten zien

dat enige voorzichtigheid bij de interpretatie van resultaten verkregen bij gebruik

van moderne analyseapparatuur op zijn plaats is.

12

Publications

The publications designated 2, I, 5 and 11, which are incorporated in this thesis as Chapters II. III. IV and VII respectively, are reprinted with permission from the relevant Journals:

1. Hydrazone formation of 2,4-dinitrophenylhydrazine with pyrroloquinoline qui­ none in porcine kidney diamine oxidase. R.A. van der Meer, J.A. Jongejan, J. Frank, jzn. and J.A. Duine. FEBS Letters (1986) 206, 111-114.

2. Methylamine oxidase from Arthrobacter PI: A bacterial copper-quinoprotein amine oxidase. J. van Iersel, R.A. van der Meer and J.A. Duine. Eur. J. Bio-chem. (1986) 161, 415-419.

3. A vitamin in disguise? J.A. Jongejan, R.A. van der Meer and J.A. Duine. Trends Biochem. Sci. (1986) //, 511.

4. Covalently bound pyrroloquinoline quinone is the organic prosthetic group in human placenta! lysyl oxidase. R.A. van der Meer and J.A. Duine. Biochem. J. (1986) 239, 789-791.

5. Phenylhydrazine as probe for cofactor identification in amine oxidoreductases: Evidence for PQQ as the cofactor in methylamine dehydrogenase. R.A. van der Meer, J.A. Jongejan and J.A. Duine. FEBS Lett. (1987) 221, 299-304.

6. Copper-containing amine oxidases (EC 1.4.3.6) have covalently bound PQQ and not PLP as organic cofactor. J.A. Duine, J.A. Jongejan and R.A. van der Meer (1987) in: Biochemistry of Vitamin B6. Proceedings of the 7th international congress on chemical and biological aspects of vitamin B6 catalysis (T. Kor­ pela and P. Christen eds.) Birkhauser Verlag, Basel pp. 243-252.

7. Phenylhydrazines as probes for cofactor identification and for interactions in copper-quinoprotein amine oxidases. R.A. van der Meer, J.A. Jongejan and J.A. Duine. Rec. Trav. Chim. Pays-Bas (1987) 106, 364.

8. Spectrophotometric studies on pyrroloquinoline quinone-copper(ll) complexes as possible models for copper-quinoprotein amine oxidases. J.A. Jongejan, R.A. van der Meer, G.A. van Zuylen and J.A. Duine. Rec. Trav. Chim. Pays-Bas (1987) 106. 365.

9. Dopamine /3-hydroxylase from bovine adrenal medulla contains covalently-bound pyrroloquinoline quinone. R.A. van der Meer, J.A. Jongejan and J.A. Duine. FEBS Lett. (1988) 231, 303-307.

10. Pyrroloquinoline quinone (PQQ) is the organic cofactor in soybean lipoxyge-nase-1. R.A. van der Meer and J.A. Duine. FEBS Lett. (1988) 235, 194-200. 11. Evidence for PQQ as cofactor in 3,4-dihydroxyphenylalanine (dopa)

decarboxy-lase of pig kidney. B.W. Groen, R.A. van der Meer and J.A. Duine. FEBS Lett. (1988) 237, 98-102.

12. Identification and quantification of PQQ. R.A. van der Meer, J.A. Jongejan and J.A. Duine (1989) in: Proceedings of the first international symposium on PQQ and quinoproteins (J.A. Jongejan and J.A. Duine, e d s ) . Kluwer Academic Press,

13. Primary s t r u c t u r e of a PQQ-containing peptide f r o m porcine kidne> diamine oxidase. R.A. van der Meer, P.D. van Wassenaar, J.H. van Brouwershaven and J.A. Duine (1989) i n : Proceedings o f the f i r s t international symposium on PQQ and q u i n o p r o t e i n s (J.A. Jongejan and J.A. Duine, eds.). K l u w e r Academic Press, Dordrecht, pp. 348-350.

14. Pyrroloquinoline quinone (PQQ) as c o f a c t o r in galactose oxidase (EC 1.1.3.9). R.A. van der Meer. J.A. Jongejan and J.A. Duine. J. Biol. Chem. (1989) 264, in press.

15. On the biosynthesis of free and covalently bound PQQ: Glutamic acid decar-boxylase f r o m Escherichia coll is a p y r i d o x o - q u i n o p r o t e i n . R.A. van der Meer. B.W. Groen and J.A. Duine. FEBS Lett. (1989). accepted f o r p u b l i c a t i o n .

!(>. Primary s t r u c t u r e o f a pyrroloquinoline quinone (PQQ) containing peptide i s o ­ lated f r o m porcine kidney diamine oxidase. R.A. van der Meer. P.D. van W a s ­ senaar, J.H. van Brouwershaven and J.A. Duine. Biochem. Biophys. Res. C o m m . (1989) 159, 726-733.

17. Analysis of quinoproteins w i t h covalently bound c o f a c t o r and p y r r o l o q u i n o l i n e quinone (PQQ) derivatives. R.A. van der Meer. A.C. Mulder. J.A. Jongejan and J.A. Duine. Manuscript in preparation.

Contents

Abbreviations 3 Chapter I: General Introduction and Summary

1. Identification of cofactors in enzymes 5

1.1 Introduction 5 1.2 Experimental approaches 5

1.2.1 Non-covalently bound cofactors 6 1.2.2 Covalently bound cofactors 8 1.3 Covalently bound cofactors: selected examples 8

1.3.1 Covalently bound flavins 8 1.3.2 Covalently bound biotin 12 2. Pyrroloquinoline quinone (PQQ) 14 2.1 Introduction 14 2.2 Physical properties 15 2.3 Chemical properties 16 2.4 Biological effects 18 2.5 Biosynthesis 18 2.6 Analysis 19

2.6.1 High performance liquid chromatography methods 19

2.6.2 Biological assays 19 3. Identification and quantification of covalently bound PQQ 20

3.1 Introduction 20 3.2 The hydrazine method 21

3.3 The hexanol extraction procedure 24 3.4 Conclusions, implications and perspectives 26

Chapter II: Methylamine oxidase from Arthrobacter PI: A bacterial

cop-per-quinoprotein amine oxidase 29

Chapter III: Hydrazone formation of 2,4-dinitrophenylhydrazine with pyr­

roloquinoline quinone in porcine kidney diamine oxidase 39

Chapter IV: Phenylhydrazine as probe for cofactor identification in amine oxidoreductases: Evidence for PQQ as the cofactor in me­

thylamine dehydrogenase 45

Chapter V: Primary structure of a pyrroloquinoline quinone (PQQ) con­ taining peptide isolated from porcine kidney diamine oxidase 53 Chapter VI: Evidence for PQQ as cofactor in

3,4-dihydroxyphenylaIa-nine (dopa) decarboxylase of pig kidney i.l

Chapter VII: Pyrroloquinoline quinone (PQQ) as cofactor in galactose

C h a p t e r V I I I : O n t h e b i o s y n t h e s i s o f f r e e a n d c o v a l e n t l y b o u n d P Q Q : G l u t a m i c a c i d d e c a r b o x y l a s e f r o m Escherichia coli i s a p y r i -d o x o - q u i n o p r o t e i n 75 C h a p t e r I X : A n a l y s i s o f q u i n o p r o t e i n s w i t h c o v a l e n t l y b o u n d c o f a c t o r a n d p y r r o l o q u i n o l i n e q u i n o n e ( P Q Q ) d e r i v a t i v e s 81 S a m e n v a t t i n g 9S R e f e r e n c e s 99 C u r r i c u l u m v i t a e 108

A b b r e v i a t i o n s Ala Arg Asn Asp ATP BSA BSAO C o n A DEAE D M A M B DNPH EC EDC ENDOR ESR FAD FMN FPLC G A O G A O , GAOc GAO Gly HABA His h . p . l . c . lie Lys MADH MeAO MH N A D * NADH NADP* NADPH NMR N o r - l e u PEG PH Phe PITC PKDAO PLP ' i n r e d a l a n i n e a r g i n i n e a s p a r a g i n e a s p a r t i c acid a d e n o s i n e 5 ' - t r i p h o s p h a t e b o v i n e s e r u m a l b u m i n e b o v i n e s e r u m a m i n e o x i d a s e C o n c a n a v a l i n e A d i e t h y l a m i n o e t h y l p - d i m e t h y l a m i n o m e t h y i b e n z y l a m i n e 2 , 4 - d i n i t r o p h e n y i h y d r a z i n e E n z y m e C o m m i s s i o n N - e t h y l - A T - ( 3 - d i m e t h y l a m i n o p r o p y l ) c a r b o d i i m i d e . H C l e l e c t r o n n u c l e a r d o u b l e r e s o n a n c e e l e c t r o n s p i n r e s o n a n c e flavin a d e n i n e d i n u c l e o t i d e (oxidized f o r m ) flavin m o n o n u c l e o t i d e (oxidized f o r m ) f a s t p e r f o r m a n c e liquid c h r o m a t o g r a p h y g a l a c t o s e o x i d a s e i n a c t i v a t e d f o r m of GAO o x i d i z e d f o r m of GAO r e d u c e d f o r m of G A O glycine 4 - h y d r o x y a z o b e n z e n e - 2 ' - c a r b o x y l i c a c i d h i s t i d i n e high p e r f o r m a n c e liquid c h r o m a t o g r a p h y i s o l e u c i n e lysine m e t h y l a m i n e d e h y d r o g e n a s e m e t h y l a m i n e o x i d a s e m e t h y l h y d r a z i n e n i c o t i n a m i d e a d e n i n e d i n u c l e o t i d e (oxidized f o r m ) n i c o t i n a m i d e a d e n i n e d i n u c l e o t i d e ( r e d u c e d f o r m ) n i c o t i n a m i d e a d e n i n e d i n u c l e o t i d e p h o s p h a t e ( o x i d i z e d f o r m ) n i c o t i n a m i d e a d e n i n e d i n u c l e o t i d e p h o s p h a t e ( r e d u c e d f o r m ) n u c l e a r m a g n e t i c r e s o n a n c e n o r l e u c i n e p o l y ( e t h y l e n e g l y c o l ) pheny I h y d r a z i n e p h e n y l a l a n i n e pheny l i s o t h i o c y a n a t e p o r c i n e k i d n e y d i a m i n e o x i d a s e p y r i d o x a l - S ' - p h o s p h a t e

PQ PQQ PQQH' PQQH2 P Q Q - H20 PQQ-PH PQQ-DNPH PTC PTH SDS S e r TFA T h r Trp T y r Val Wurster's blue

pyrroloquinoiine. semisvstematic name for 2.7.9-tricarbo\\-lr/-pyrrolo(2.3-/)quinoline

pyrroloquinoiine quinone, semisvstematic name for 2,7.')-tricarboxy-l//-pyrrolo(2,3-flquinoline—1.5-dione semiquinone form of PQQ quinol form of PQQ covalently hydrated PQQ phenylhydrazone of PQQ and PH 2,4-dinitrophenylhydrazone of PQQ and DNPH phenylthiocarbamyl phenylthiohydantoin sodium dodecyl sulphate serine trifluoracetic acid threonine tryptophane tyrosine valine

Chapter I

GENERAL INTRODUCTION AND SUMMARY

1. Identification of cofactors In enzymes

1.1 Introduction

Some enzymes depend for their activity only on their protein chain, others require one or more nonproteinaceous compounds, called cofactors. The cofactor (the term cofactor as used here encompasses the notions coenzyme and prosthet­ ic group) can either be a metal ion or an organic molecule. Cofactors are gene­ rally stable to heat, whereas most enzyme proteins are not. They are usually involved in transfer of functional groups, of specific atoms, or of electrons. Removal of the cofactor from the protein (the holo-enzyme) leads to an inactive form, the so-called apoenzyme.

The beginning of modern biochemistry was characterized by an intense level of activity directed towards cofactor chemistry and the elucidation of metabolic pathways. These investigations were remarkably successful, resulting in a (close to) full description of enzymatic cofactors and vitamin requirements in higher organisms. Among enzymatic redox reactions, it appeared that activities could be accounted for by the presence of either flavins, nicotinamides, pterins and heme or non-heme metal centers. Although cofactor research was historically focussed on vitamins, nowadays this is not the case. Recent work on the biochemistry of methanogens (anaerobic, methane-producing bacteria) and the methanotrophs (aerobic bacteria, utilizing methane as a carbon source) has thrown up several new cofactors, mostly confined to one of these specialistic groups of bacteria [Leigh and Wolfe, 1983; Anthony, 19821.

Examples of recently discovered cofactors with a more wide distribution are molybdopterin (see a recent review by [Hageman and Rajagopalan, 1986]) and the tyrosyl free radical (see a recent review by [Prince, 19881). In view of the rather large number of enzymes for which cofactor identification has not been carried out [Duine and Jongejan, 1989b], the recently discovered cofactors might even be wider distributed as presently thought, and others might have been overlooked, exemplified by PQQ (see section 2.1).

1.2 Experimental approaches

In practice, an indication for the presence of a cofactor in an enzyme can be obtained by measuring the absorption spectrum of the homogeneous enzyme p r e ­ paration. Unequivocal identification of the cofactor is. however, less simple. The reason for this is that binding of the cofactor to the protein is not without con­ sequences for its electronic transition states. Mostly, therefore, inspection of enzyme spectra does not lead to an unambiguous conclusion and extraction of

the cofactor from the enzyme is necessary to allow comparison with authentic material (in addition, to distinguish a cofactor from an allosteric modulator, fur­ ther experiments are necessary). However, cofactors can be either attached to the protein chain with non-covalent or with covalent bonds. It will be clear that special procedures have to be used in the latter case.

1.2.1 Non-covalently bound cofactors

For the determination of non-covalently bound cofactors, numerous methods have been developed: biological (using either t e s t animals, microorganisms, or apo-enzymes). chemical, and a great diversity of other methods. Many of these methods have gradually been abandoned and will therefore not be discussed in detail here. For details on these abandoned methods, the reader mat be referred to reviews on this subject CMickelsen and Yamamoto, 1958: Steyn-Parve and Mon-foort, 1963; Lambooy, 1963; Arroyave and Bressani, 19631.

(a) Assays with test animals

Biological assays using animals were the first to be developed. They were the onlj practicable methods in the days before the cofactor structures were known. and are based on the vitamin action of the cofactor in question. Generally, they are specific, but expensive in terms of time, animals, and material under assay. Therefore, most of them have been abandoned for routine use while some are occasionally employed for a specific purpose, for instance to t e s t the physiologi­ cal availability of a vitamin in certain foodstuffs and diets.

Within this group of methods, a further classification can be made, as follows: (i) protective methods, based on the prevention against a disease, e.g. polyneuritis prevented by thiamine administration; (ii) growth methods, based on the stimula­ tion of growth rate of young animals (this method is not ver> specific so t h a t precautions must be taken to ensure that the growth response measured derives solely from the vitamin or cofactor under investigation); (iii) curative methods (animals that have developed a certain disease are treated with the vitamin under consideration, and the "length of the curing period" or the "percentage of cured animals" during a number of days, is taken as a measure of the amount of vita­ min).

(bl Microbiological methods

Most methods for the determination of cofactors and vitamins employing mi­ croorganisms measure the growth rate of the latter in terms of turbidity of the culture. These methods are still used fairly frequently because they are sensitive, relatively rapid, and inexpensive (when compared to the aforementioned assay methods using test animals). However, some of the microorganisms used show a certain lack of specificity: for example with the assay of thiamine as a growth factor CSarett and Cheldelin, 1944: Deibel e£ a/.. 1957], the requirement for thia­ mine can also be met by the pyrimidine and thiazole moieties of the molecule, either as the single compound or in combination. Similarly, for the assay of

lipoic acid, the requirement of specific E. coli mutants for lipoic acid can be spared or replaced by acetate plus succinate or lysine plus methionine [Herbert and Guest, 19701. A well known example of a microbiological method which is still used today is the assay of the vitamin B6 group, as described by Haskell

and Snell [19701.

(c) Chemical methods

Chemical methods are the ones which are the most widely used for routine d e ­ terminations. They have a relatively high specificity, and they are rapid and rea­ sonably accurate (when compared to the microbiological and animal methods).

In contrast to the aforementioned methods, most of the chemical methods need a pretreatment of the material under assay for the following reasons:

(i) The cofactor must be dissociated from the material. Common methods are heating to 100 °C in dilute acid (at pH 2 to 3); extraction using an organic s o l ­ vent, e.g. chloroform, methanol or ethanol (either boiling or not, depending on the strength of the binding between cofactor and protein); or alkaline extraction using e.g. 0.5 M NaOH.

(ii) If the cofactor is also present in a phosphorylated form, the phosphate esters can be hydrolyzed. This is usually performed by using a phosphatase active on the phosphorylated cofactor under investigation.

(iii) In many cases the cofactor has to be separated from interfering substances a n d / o r concentrated by an adsorption and elution step (this may also be neces­ sary for the aforementioned methods).

An example which is still in use today is the determination of thiamine and its derivatives via the thiochrome method. This method is based on the observation by Barger et al. [19351 that oxidation with alkaline ferricyanide converts thiamine into a compound with intense blue fluorescence, thiochrome. The oxidation step is carried out on an extract of a thiamine containing sample prepared by heating it with dilute acid at 100 °C, followed by hydrolysis of thiamine-phosphate esters (with phosphatase) and adsorption and elution of thiamine from bentonite [Jan-sen, 19361. The thiochrome formed is extracted into isobutanol and the fluore­ scence of the extract is measured. This method allows determination of esterified as well as free thiamine by running two determinations, one with and one wi­ thout the phosphatase treatment (the fluorescent products formed from the thia­ mine phosphates are only slightly soluble in isobutanol).

(d) Enzymological methods

Enzymological methods are either based on the use of an apoenzyme for the cofactor of interest or on an enzymatic reaction using the cofactor in a non-ca­ talytic way, that is as a component in the reaction. The assays are characterized by a very high specificity and a high sensitivity the latter requiring special p r e ­ cautions in avoiding contaminations. An example of an assay where the cofactor as a component of the reaction is the assay for Coenzyme A (CoA), based on the observation that the arsenolytic decomposition of acetyl phosphate is CoA depen­ dent in the presence of phosphotransacetylase. With this assay, the residual

amount of acetyl phosphate is determined chemically [Stadtman and Kornberg, 1953]. An example of an assay using an apoenzyme is the determination of pyri-doxal phosphate with tryptophanase in the apo-form [Wada et al., 1957].

Most of these enzymological methods also require a pretreatment of the sample, as described for the chemical methods.

(e) Other methods

Several other methods have been worked out using techniques like chromato-graphy/h.p.l.c. (with a diversity of chromatography materials), gas chromatogra-phy, electrophoresis, and labelling with radioactive compounds. Because these are based on broadly applicable and well described analytical procedures, no overview will be given here.

1.2.2 Covaiently bound cofactors

The determination of covaiently bound cofactors and its mode of binding is a far more difficult area to address experimentally when compared to that of non-covalently bound cofactors. However, basically the same methods as d e ­ scribed for non-covalently bound cofactors can be applied after the cofactor in question has been obtained in a free form.

Detachment of a covaiently bound cofactor can be achieved by proteolytic dige­ stion of the enzyme (see the flavin-containing enzymes, section 1.3.1), or by acid hydrolysis (see the biotin and lipoic acid containing enzymes, section 1.3.2). 1.3 Covaiently bound cofactors: selected examples

In this section, studies on identification and linkage of a selection of cofactors will be discussed. The examples should be seen merely as an illustration of the different approaches used.

1.3.1 Covaiently bound flavins

Occurrence

The first covaiently bound flavin to be discovered was the flavin component of mammalian succinate dehydrogenase [Kearney & Singer. 1955; Singer et al.. 1956]. Proof that its structure is 8a-M3)-histidyl-FAD (Fig.1) came 15 years later [Wal­ ker & Singer, 19701. At the moment, five different structures in this group of enzymes have been identified and chemically synthesized. These structures involve attachment of amino acid residues to either the 8ar-methyl group or the C(6) of the aromatic ring of the flavin. Already more than 25 different enzymes have been shown to contain covaiently linked flavin, enzymes originating from bacteria as well as higher vertebrates. Examples for each of the structures identified so far are given in Table 1.

H

Nr^

"

N-H

C

5 41

C H .

H2N - C H - C O O H

Figure li Structure of 8ar-Aft3)-hlatldyl-FAD. R denote» the nmilnlDg part of the FAD molecule.

Tabla 1: Enxymaa containing covalently bound flavin.

Enzyme Source Reference

8or-Ml)-Hlatldyl-FAD /3-Cyclopiazonate oxidocyclase L-Gulono-Y-lactone oxidase Cholesterol oxidase Penicillium cyclopium Mammalian liver Schizophyllum commune

Kenney & Singer, 1977 Kenney et a I., 1976 Kenney et a/., 1979 8<r-M3)-Hlatldyl-FAD Succinate dehydrogenase Choline oxidase Fumarate reductase Saccharomyces cerevisiae Arthrobacter globiformis Escherichia coli

6-Hydroxy-D-nicotine oxidase Arthrobacter oxidans

Oestreicher et al., 1980 Onishi & Yagi. 1979 Weiner & Dickie, 1979

Mohler et al., 1972

D-Gluconate dehydrogenase Pseudomonas fluorescens Mclntire et .?/.. 1985

8<r-£-cyatelnyl-FAD Monoamine oxidase A Monoamine oxidase B Sarcosine oxidase3 Human placenta Bovine liver Corynebacterium

Salach & Detmer. 1979 Walker et a/., 1971 Hayashi et al., 1980

Bat- O-tyroay 1-FAD

p-Cresol methylhydroxylase Ps. putida M c l n t i r e et a/., 1981

6-S-cyatelnyl-FMN Trimethylamine dehydrogenase Trimethylamine dehydrogenase Hyphomicrobium X Bacterium W3A1 Steenkamp, 1979 Ghisla et a/., 1980 Proteina with covalently bound flavin of unknown linkage

D-Sorbitol dehydrogenase Gluconobacter

melanogenus

Ameyama & Shinagawa, 1984

"Reported to contain one mol each of covalently and non-covalently bound flavin per mol of enzyme.

The identification of covalently bound flavin in an enzyme has become a rela­ tively simple task. The conventional way is to precipitate and wash the protein with trichloroacetic acid (to remove non-covalently bound flavins), digest it for a few hours with proteolytic enzymes (usually trypsin and chymotrypsin). and look for flavin-type fluorescence. Use is made of the differences in the quantum yield of fluorescence for certain flavin-adducts between pH < 3.5 and neutral pH and in the enhancement of fluorescence on oxidation with cold performic acid (the latter treatment is necessary to overcome the very intense internal quenching of fluorescence in thio ether-linked and in tyrosyl-linked flavins). 6-S-Cysteinylflavin is distinguished from other covalently bound flavins by its abnormal absorption spectrum, while Sa-O-tyrosylflavin becomes fluorescent only after reductive cleavage with dithionite and reoxidation by air. Another method to detect cova­ lently bound flavin is to subject the native protein to polyacrylamide gel electro-phoresis under denaturing conditions. This separates free flavins from cova­ lently bound flavins. The latter can be detected by their yellowish fluorescence (if required, low pH and spraying the gels with performic acid is possible).

Comparison of the amino acid sequences around the flavin reveals relatively little homology among enzymes containing the same type of flavin structure [Singer & Mclntire. 1984]. except between enzymes of closely related function (e.g. mammalian succinate dehydrogenase and bacterial fumarate reductase). An­ other exception is the couple bacterial choline oxidase/6-hydroxy-D-nicotine oxi-dase [Bruhmuller & Decker. 1973].

Analysis

Because of the different modes of attachment of flavin to biological material, no straightforward approach for all cases is available. One commonly s t a r t s with digestion of the denatured and well washed protein (to remove non-covalently bound flavin) using proteolytic enzymes, yielding a flavin containing peptide. After this proteolytic digestion it is essential to purify the flavin peptide first in order to remove materials which interfere in subsequent tests (e.g. hemes origi­ nating from cytochromes). Most commonly, a Florisil column is used for that purpose. The flavin-peptide is adsorbed, washed extensively, and eluted with 5 % (v/v) pyridine. In order to eliminate losses by having the flavin present in several phosphorylated forms (which could result from breakdown during manipulation), it is desirable to convert the flavin in the peptide to its dephosphorylated form at this point. This can be achieved by sequential treatment with nucleotide pyro-phosphatase and alkaline pyro-phosphatase. The peptide is then purified on thin-layer cellulose plates.

While the flavin peptide is seldom pure at this point it may be sufficiently pure for unambiguous identification of the type of attachment of the flavin. For this purpose the peptide is digested using proteolytic enzymes to liberate all but the flavin-linked amino acid (which resists digestion by most peptidases). and the flavin-amino acid derivative (aminoacyl flavin) is then further purified by ion-exchange chromatography, h.p.l.c. thin-layer chromatography (TLC). or high-vol­ tage electrophoresis. These procedures can already identify the type of a t t a c h

-ment of the flavin by comparison with synthetic standards.

Quantitative determination of the covalently bound flavin of a pure protein presupposes knowledge of the structure involved. Provided that no other chromo-phore is present in the enzyme, the flavin content is calculated from the absor-bance at 450 nm. With 6-S-cysteiny I flavin this method is ruled out because of the anomalous absorption spectrum.

A more sensitive procedure for the quantification of histidylflavins is based on the difference in fluorescence of the aminoacyl flavin between pH 3.2 and 7. De­ tails of this assay are described by Singer et al.. 1984 and Singer and Edmondson, 1980. A rather satisfactory method for the quantitative analysis of tyrosylflavin is based on the reductive cleavage of the flavin from the denatured protein and measurement of its fluorescence [Mclntire et al.. 1981].

To ilustrate how the elucidation of flavin binding has been tackled, the case of monoamine oxidase will be discussed. Monoamine oxidase was purified and di­ gested with trypsin and chymotrypsin to yield peptides containing covalently bound flavin [Kearney et al., 19711. Subsequently, a series of chromatographic procedures was applied yielding a pure pentapeptide of riboflavin-5'-phosphate. Edman degradation, followed by dansylation, revealed the amino acid sequence: Ser-Gly-Gly-X-Tyr, X being the amino acid to which the flavin is attached. The involvement of the 8tr-carbon of the riboflavin in the binding with the amino acid was concluded on the basis of analysis of the optical spectra in the neutral and cationic forms of the flavin and of the ESR spectrum of the free radical cation. [Kearney et al., 19713. The amino acid to which the flavin is bound was identified as cysteine after acid hydrolysis of performic acid oxidized peptide. The linkage of this cysteine to the flavin was identified as a thioether [Kearney et al., 1971; Walker et al., 1971]. Evidence for this binding of cysteine via a thioether a t the 8 a - C H3 group of the flavin was obtained by comparison of the UV/VIS and ESR

spectra, chemical stability, and fluorescence characteristics of the flavin peptide isolated from the enzyme with the properties of synthetic 8a-S-cysteinylriboflavin [Walker et al., 1971 ].

The process of attachment of flavin to the apo-enzyme

Despite the importance of this group of flavoproteins and major efforts in s e ­ veral laboratories, very little is known about the mechanism by which FMN or FAD is inserted into covalent linkage during the assembly of these enzymes. Even the fundamental question whether the attachment of the flavin to the protein is an enzymatic process or whether the structure of the assembled polypeptide chain predisposes it to facile binding of the flavin in covalent linkage has been a long standing problem [Walsh, 1981]. However, recent studies by the group of Brandsch [Brandsch and Bichler, 1987 and Nagursky et al., 1988] on 6-hydroxy-D-nicotine oxidase showed that covalent flavinylation of apo-6-HDNO requires, besides FAD, phosphoenolpyruvate, while no phosphorylated flavin intermediate could be detected in the flavinilation reaction, and no requirements for a synthe-tase activity was found. No flavin (riboflavin, FMN, or FAD) activated in the 8a

p o s i t i o n has been found in preparations actively biosynthesizing enz\mes which contain covalently bound f l a v i n .

1.3.2 Covalent binding of blotln

Occurrence

The enzymatic role o f biotin as a covalently bound " C Oz carrier" is now u n ­ equivocally established. Insight i n t o the mechanism o f action was gained f o l l o w ­ ing over a h a l f - c e n t u r y of n u t r i t i o n a l , chemical, and metabolic studies, commenc­ ing a t the end o f the nineteenth century w i t h the d e m o n s t r a t i o n of the toxic properties of raw egg w h i t e , later shown to be due t o avidin, a g l y c o p r o t e i n w i t h an extraordinary a f f i n i t y f o r b i o t i n . The correct s t r u c t u r e o f b i o t i n (Fig.2) was determined by the g r o u p o f Du Vigneaud [ M e l v i l l e et al., I942J, which was later c o n f i r m e d by chemical synthesis [ H a r r i s et al., 1945] and X - r a y crystallography [ T r o t t e r and H a m i l t o n . I960]. By 1950, biotin had been implicated in a number of seemingly unrelated enzymatic processes, including (a) the decarboxylation o f oxaloacetate and succinate [Lardy et al., 1947], (b) the " W o o d - W e r k m a n reaction". t h a t is. the /3-carboxylation o f pyruvate [Wessman and Werkman, 1950], (c) the biosynthesis o f aspartate [ A h m a d and Rose, 1962], (d) the biosynthesis o f u n s a t u -rated f a t t y acids [Mager et al., 1954], and (e) the deamination o f certain amino acids [Lichstein and C h r i s t m a n , 1949]. In every instance the metabolic basis f o r these observations can now be explained in terms o f the role o f b i o t i n as an enzymatic " C 02 carrier". 0 H

N A N

Figure 2: Structure of (♦)bloUn ( 2 ' k e t o 3 , 4 l m l d a i o i l d o -2-Uttr«hydrothloph»no-n-valerlc acid.

Comparison o f t h e amino acid sequences around the b i o t i n a t t a c h m e n t s i t e r e ­ veals t h a t there is a remarkable degree o f conservation o f sequence, especially in the region immediately N - t e r m i n a l t o the biotin residues [ R y l a t t et at.. 1977). This implies t h a t these particular amino acid side chains p e r f o r m a c r i t i c a l s t r u c t u r a l or catalytic role.

Identification and quantification

Early investigations already indicated that biotin was probably covalently bound in biological materials. The i s o l a t i o n of biocytin f r o m yeast e x t r a c t b> W r i g h t et

al. [1952] and the subsequent determination o f its s t r u c t u r e as f N b i o t i n y l L l y

-sine by degradation [ K o s o w and Lane, 1962] and synthesis f r o m biotin acid c h l o ­ ride and L-lysine [Gregolin et al., 1968], suggested t h a t the site o f a t t a c h m e n t m i g h t be a lysyl residue on the enzyme. Following the incubation of l 4C - b i o t i n w i t h the propionyl CoA holocarboxylase-synthesizing system, the l 4C h o l o c a r -boxylase was p a r t i a l l y p u r i f i e d and then subjected to p r o t e o l y t i c digestion w i t h pronase [ N o m o t o et al.. I960]. The sole ' * C - l a b e l e d derivative i n the pronase digest was isolated and subsequently identified as biocytin [ K o s o w and Lane, 1962] by comparison w i t h the synthetically prepared compound.

I d e n t i f i c a t i o n o f a b i o t i n containing enzyme has become a relatively simple task. The conventional way is applying a typical i s o l a t i o n technique like c a t i o n exchange chromatography w i t h gradient e l u t i o n (pH 4.5 t o 6.5) t o an acid h y d r o -lysate o f the enzyme. Once isolated, b i o t i n and its analogues can be d i f f e r ­ entiated on paper or TLC sheets in various solvent systems [Lee et al., 1972; Ruis

et al., 1968] and quantitated at the microgram level using the c o l o r i m e t r i c reac­

t i o n w i t h p-dimethylaminocinnamaldehyde [ M c C o r m i c k and Roth, 1983]. B i o t i n and i t s analogues have also been separated by C1 8 reversed-phase chromatography once they are derivatized t o esters using p-bromophenacyl bromide f o r UV detec­ t i o n and 4 - b r o m o m e t h y l m e t h o x y c o u m a r i n f o r f l u o r i m e t r i c detection [Desbene et

al., 1983], More d i r e c t methods are: (a) h.p.l.c. o f underivatized b i o t i n and i t s

analogues, using a reversed phase C ,8 c o l u m n and m o n i t o r i n g the absorbance at 220 nm [ B o w e r s - K o m r o et al., 1986], or (b) measuring the enhancement of the fluorescence o f the binding o f biotin or its analogues, t o f l u o r e s c e i n - l a b e l l e d avidin [Nargessi and S m i t h . 1986]; (c) a s p e c t r o p h o t o m e t r i c method [Green, 1970] involving biotin displacement o f a dye (4-hydroxyazobenzene-2'-carboxylic acid (HABA)) f r o m the avidin binding site, accompanied by a spectral change at 500 nm. HABA is not bound by the avidin-biotin complex, and since the dissociation constant o f the l a t t e r is so low U0~' M) the dye is s t o i c h i o m e t r i c a l l y displaced by b i o t i n .

W i t h the recognition of the fundamental importance o f biotin i n cell m e t a b o ­ l i s m , it became necessary to obtain i n f o r m a t i o n as to the d i s t r i b u t i o n of t h i s c o -f a c t o r in nature. As most o-f the biotin present in tissues occurs in a bound f o r m , detachment was necessary. Careful and systematic investigations on the l i ­ beration o f b i o t i n demonstrated that a l m o s t complete detachment can be achieved by acid hydrolysis (4-6 M s u l f u r i c or hydrochloric acid f o r t w o hours at 120 °C) or enzymatic digestion [Thompson et al.. 194U.

Because i t is now w e l l established t h a t most, if not a l l , biotin-dependent enzymes contain b i o t i n covalently bound to a lysine residue, qualitative i d e n t i f i c a ­ t i o n as w e l l as assay o f b i o t i n f r o m biotin-dependent enzymes has become r e l a ­ tively easy. Once b i o t i n is obtained in a free f o r m (after acid hydrolysis, or p r o ­ t e o l y t i c digestion), it can be assayed using one of the aforementioned methods.

The process of attachment of biotin to the apoenzyme

The terminal step in the synthesis of b i o t i n - c o n t a i n i n g enzymes appears t o be the covalent attachment of the c o f a c t o r t o the apoenzyme, catalyzed by a s y n

-thetase requiring ATP and Mg . The type of linkage found for propionyl CoA carboxylase (biotine attached to the f-NH0 group of a lysine, see above) has also

been detected in several other biotin-containing enzymes [Allen et al., 1964; Kazi-ro et al., I96S; Wood et al., 1963]. This mode of attachment extends also to lipo-ic acid, whlipo-ich is linked through its carboxyl group in an amide linkage to a lysyl f-amino group of e.g. pyruvate dehydrogenase [Reed et al.. 1958 and Reed. I960]. 2. Pyrroloquinollne quinone (PQQ)

2.1 Introduction

In the past decade it has become clear that in addition to the well-known classes of enzymes, there is another class, the so-called quinoproteins, in which pyrroloquinoline quinone (PQQ) (Fig.3) is involved as the cofactor.

HOOC

HOOC

COOH

Figure 3: Structure of pyrroloquinollne

pyrrolo C2,3-f]-qulnoUn«-4,S-dlone). quinone (2,7.9-tricarboxy-lH-Forrest and coworkers [Salisbury et al., 1979] elucidated the structure of an acetone adduct of the cofactor and proposed a structure for the cofactor itself (methoxatin). The structure of the isolated cofactor itself was determined and evidence for the o-quinone structural element in the molecule was presented by Duine and coworkers [Duine et al., 1980]. Based on its chemical name, 2,7,9-tri-carboxy—lW-pyrrolo [2,3-/"]-quinoline-4,5-dione, the semisystematic name pyrrolo­ quinoline quinone was introduced. This name can be abbreviated to PQQ while biological relevant redox forms can be indicated as PQQH' (semiquinone form of PQQ) and PQQH2 (quinol form) (Fig. 4). In analogy with flavoproteins.

heamo-proteins, etc., PQQ-containing enzymes are called quinoproteins.

After the finding of PQQ in several bacterial dehydrogenases, it was attempted to detect this cofactor in eukaryotic enzymes, which was in fact the beginning of the research presented in this thesis.

COOH H COOH u ^COOH HOOC N ff HOOC

HOOC ^ N - ^ ^ v a - ^ ^ O H O O C ' " " N ^ ' N f OH HO O C

POO POOH' POOH2

Figure 4 : Structure of the b i o l o g i c a l relevant r e d o x form» o f PQQ: PQQH* (eemlqulnone form) and P Q Q H2 (quinol f o r m ) .

2.2 Physical properties

A b s o r p t i o n spectrophotometry, fluorescence spectroscopy and NMR s p e c t r o s c o -py of aqueous PQQ solutions show the presence of t w o i n t e r c o n v e r t i n g species. The explanation f o r this phenomenon is that there exists an e q u i l i b r i u m between unhydrated and covaiently hydrated PQQ ( P Q Q - H20 ) . due to the reactivity o f the

C5 carbonyl group o f PQQ towards nucleophiles. leading t o the hydrate (and also

to adduct f o r m a t i o n w i t h amino groups, thiol groups, etc.).

A b s o r p t i o n spectra o f PQQ at high temperatures or in organic s o l v e n t s are representative to t h a t o f unhydrated PQQ. while spectra at low temperature or o f PQQ complexed w i t h metal ions [Jongejan et al., I9873 are representative f o r t h a t o f P Q Q - H20 [Dekker et al., 19821. The absorption spectra o f PQQ (actually the sum o f PQQ and P Q Q - H20 ) and P Q Q H2 are shown in Fig. S (at pH 7.0). They are characterized by maxima at 24-9, 323 and 475 nm (S = 25400, 9898 and 742

M - ' . c i r T1 respectively) f o r PQQ, and by a maximum at 302 nm ( £3 0 2 = 30S00 V r ' . c n T1) f o r PQQH.,. The l a t t e r maximum s h i f t s t o 317 nm at pH 9.5 and 315 nm at pH 1.5 [Duine et al., 1981]. A u t o - o x i d a t i o n of P Q Q H2 readily occurs in w a t e r - c o n t a i n i n g solvents above pH 4. The absorption spectrum of P Q Q H ' has been determined by Faraggi et al. [1986], and by Duine et al. [1981]. The l a t t e r was obtained after mixing equimolar amounts o f the quinone and quinol anaero-bically in a methanol-containing LiOH s o l u t i o n (pH 12.7). This absorption spec­ t r u m o f P Q Q H ' at p H 12.7 shows a maximum at 458 nm [Duine et al., 1981], while Faraggi found a maximum at 460 nm ( w i t h a f o f 3200 M " ' . c m " ' ) a t pH 7.3 [Faraggi et al.. 1986].

The fluorescing properties o f PQQ in aqueous s o l u t i o n are due to P Q Q - H20 , since unhydrated PQQ does not show fluorescence [Dekker et al., 1982]. In agree­ ment w i t h t h i s , the fluorescence excitation spectrum shows e x c i t a t i o n maxima at 325 and 360 nm, similar t o the calculated absorption maxima o f P Q Q - H20 (249, 325 and 360 nm) (Dekker et al., 19821.

The most salient feature o f the ' H - N M R spectrum o f PQQ is the low f i e l d p o ­ sition of the p y r r o l o N - H signal (13.3 ppm), which is caused by the s t r o n g i n t e r ­ action between this hydrogen and the C9 carboxylic acid group. The C3~H and

C8- H signals are found at approximately 7.4 and 8.6 ppm, respectively [Duine e t al., 1979. 1980 and 1981].

Using 99 % U-, 3C-labelled PQQ. all signals of the , 3C-NMR spectrum of PQQ

could be assigned [Frank et al., 1989: Unkefer et al., 1989].

0.4

-

0.3

-

0.2

-

0.1

200 300 400 500

Wavelength (nm)

0

600

700

Figure S: Absorption apactra of PQQ and PQQH2 PQQ was dissolved

in O.OS M potassium phosphate pH 7.O. and the absorption spectrum measured before ("" ~) and after reduction (~ -) w i t h H , _{in the} presence of PtO~ (reproduced from LDuine et a/.. 1981]).

2.3 Chemical properties

An interesting feature of PQQ is its remarkable stability towards agressive acids and bases, as compared to the more labile cofactors such as NAD and flavins. PQQ is stable in hot concentrated H2S 04, HCI, as well as in 1 M NaOH.

However, using 10 M NaOH [Lobenstein-Verbeek et al., 1984] or mixtures of H N 03 and H2S 04, it is degraded to biologically inactive compounds (J. Frank.

personal communication).

Reduction of PQQ to its quinol form can be easily achieved by a variety of agents, such as hydrazines. NaBH4, /3-mercaptoethanol and cysteine [Duine et at.,

1981; Itoh et al., 1986b). These reduction agents are more rapidly oxidized by PQQ than by certain artificial electron acceptors. The kinetics of the oxidation of free

thiol groups [Itoh et al., 1986a], NADH and oxidation of PQQH2 by molecular

oxygen tltoh et al., 1986b] have been described.

Although PQQ seems very suited for ligation of metal ions, so far only prelimi­ nary investigations have been published. Titration experiments

performed with Cu2* and PQQH2 [Jongejan et al., 1987] indicated that the redox

reaction proceeds very rapidly. Similar experiments with PQQ showed a preference of Cu2* for complexation with PQQ-H20, suggesting involvement of the N(6) and

C5= 0 sites [Jongejan et al., 1987]. On the other hand, in the ternary complex

between PQQ, Cu(II) and bipyridine, the Cu-ion is in the equatorial plane with the N(6) and 7-COOH sites of PQQ [Suzuki et al., 1987], preventing the accessi­ bility of the o-quinone moiety of PQQ. in the ternary complex of PQQ, Cu(II) and terpyridine, the Cu ion is bound via the N(6) and 7-COOH, in the equatorial and axial directions, respectively. In this situation, the aromatic planes of the Cu(II) complex of terpyridine and that of PQQ are perpendicular to each other, so that the o-quinone moiety of PQQ is easily accessible.

The already mentioned reactivity of PQQ towards nucleophiles can lead t o adduct formation. If these adducts also exist in quinoproteins, this provides an explanation for the variety of absorption spectra seen among quinoproteins and the differences with respect to PQQ. Although these adducts may be stable in an enzyme, model studies performed by van Kleef [van Kleef et al., 1988] have shown that adducts of PQQ and ami no acids are not. The adducts decompose under formation of the corresponding aldehyde, C Oz and a reduced form of PQQ

[Itoh et al., 1986b]. The latter becomes reoxidized under aerobic conditions so that a cyclic process takes place with catalytic amounts of PQQ. In addition, depending on the amino acid used, formation of an oxazole (Fig.6) can occur (a dead-end product), with a concomitant loss of PQQ (as measured with a biologi­ cal assay). Although no systematic studies have been performed on the occur­ rence of these oxazoies in biological samples, it can be expected that free PQQ will only exist under rather clean conditions, e.g. in the culture fluid of methy-lotrophic bacteria, grown on a mineral medium with alcohols as carbon and ener­ gy source.

HOOC

N

,COOH

HOOC

2.4 Biological effects

In view of the widespread occurrence of PQQ. it could be expected that it has biological significance, either as a growth factor for microorganisms a n d / o r as a vitamin in mammalian organisms. In this context it is interesting to mention that many bacteria produce the protein part of quinoproteins but not the cofactor [Duine et al-, 19861. One striking example is E. coli where the well known labo­ ratory strains all contain the quinoprotein glucose dehydrogenase apoenzyme [Hommes et al., 19841. Functionality can be achieved, however, as was demon­ strated for an E. coli C PTS" mutant (which is unable to metabolize glucose via the phosphorylative route) since it grew on glucose only in the presence of PQQ [Hommes et al., 19841. The fact that several other bacteria excrete PQQ into their culture medium (e.g. methylotrophs. acetic acid bacteria. Pseudomonas spe­ cies) could be interpreted as a phenomenon related to provision of cofactor to bacteria producing quinoprotein apoenzymes. Although the latter organisms do not synthesize PQQ, it should be realized that the) are unable to produce the free form but most probably synthesize the covaiently bound form (see section 2.S).

Since mammalian quinoproteins contain PQQ in a covaiently bound form, and preliminary indications [Van Kleef and Duine. 19891 on the biosynthesis in bacteria sugested that this process could occur on the enzyme itself, it seems unlikely that PQQ is a vitamin. However, in the light of the alreadj proposed role of PQQ as a modulator of connective tissue formation [Hanauske-Abel et al., 19871, and positive effects noted on administration of PQQ to the diet of rats (Matsu-moto et al., I9891, indirect effects cannot be excluded. Thus, it would be inter­ esting to trace uptake and distribution of PQQ in order to elucidate its role in mammals. In view of the reactivity of PQQ towards nucleophiles (section 2.3). it is highly improbable that the compound is available in its free (oxidized) form under in vivo conditions, where substantial concentrations of nucleophiles are normally present. However, the presence of a special carrier (preventing conver­ sion to e.g. oxazoles) or an "oxazolase" cannot be excluded at the moment [Duine and Jongejan, 1989bl.

2.S Biosynthesis

Tyrosine and glutamic acid are the precursors for biosynthesis of free PQQ in methyltrophic bacteria [van Kleef and Duine, 1988; Houck et al.. 19881. E. coli strains produce covaiently bound PQQ (in glutamic acid decarboxylase). but not the free form (Chapter VIII). The most likely explanation for this finding is that biosynthesis of the covaiently bound cofactor does not s t a r t with free PQQ but proceeds in situ. Since production of glucose deh>drogenase holo-enzyme in E.

coli requires incorporation of all 4 genes (cloned from Acinetobacter calcoaceti-cus LMD 79.41 [Goossen et al.. 19891). either synthesis of free PQQ occurs in a

quite different way or the 4 genes are required for processing of covaiently bound PQQ to the free form.

2.6 Analysis

Two types of analysis procedure have been developed for free PQQ: biological assays based on reconstitution of a quinoprotein apo-enzyme, and h.p.l.c. methods using reversed phase chromatography materials, providing PQQ concentrations are high and contamination is low. Details will be given in the following paragraphs.

Recently, two other approaches were reported. The method of Gallop and c o -workers (Flueckiger et al., 1988] is based on the decarboxylation activity of PQQ for glycine. Nitroblue tetrazolium is used to reoxidize PQQH2 and the formazan

formation is followed spectrophotometrically at 530 nm. Because valine does not react with PQQ, this compound is used in the control experiment. Although this method is able to detect free PQQ, adducts will not react and the effect on the assay of compounds forming condensation products with PQQ, is still unknown. Suzuki et al. [1989] have developed a method employing gas chromatography of derivatized PQQ, but also in this case similar comments can be given.

2.6.1 High performance liquid chromatography methods

PQQ, its reduced forms, and several of its derivatives have been analyzed on a C| 8 reversed phase column, using absorption or fluorescence detection [Duine

et al., 1983]. Ion-suppression chromatography with a CH3OH gradient in 0.4 %

H3P 04 gave satisfactory results for PQQ, PQQH2, and several derivatives [Duine

et al., 1983J. Ion-pairing chromatography works even better since the peak of

PQQ in the chromatogram is much sharper [Duine et al., 1983]. Fluorescence detection is preferred over absorption detection because most of the contami­ nants do not fluoresce (the latter property makes the requirements for purity of the samples less stringent). If samples are heavily contaminated (e.g. cell free extracts), it is advisable to perform a clean-up with an Amberlyst A21 anion-exchanger [van Kleef et al., 1987]. Controlled adduct formation with an aldehyde or ketone can be used to check the identity of a presumed PQQ-peak in the chromatogram [Duine et al., I9831.

2.6.2 Biological assays

This method was first performed with glucose dehydrogenase apoenzyme from

Acinetobacter calcoaceticus [Duine et al., 1979J. Meanwhile, several naturally

occurring and self-prepared apo-quinoproteins have been used for that purpose, either in purified form, or present in membrane particles or whole cells [Hauge, 1964; Duine et al., 1979; Kilty et al., 1982; Ameyama et al., 1980; Duine et al.. 1983; van Kleef et al., 1987; Ameyama et al., 1985; Geiger and Goerisch, 1987; Ameyama

et al., 1981; Groen et al., 1986]. Factors that are important to make a choice are:

the availability, the ease of preparation, and the stability of the apo-enzyme; absence of background activity; a high turnover number; rapid reconstitution; no interference of other enzymes (especially important if unpurified apoenzymes, membrane particles or whole cells are used).

care to avoid contamination with the cofactor. Thus, it was found [van Kleef et

al., 19871 that chromatographic column materials and laboratory glassware b e ­

comes easily contaminated with PQQ, giving high backgrounds so that false positive results are obtained if no checks are made. Since PQQ is a very stable compound, glassware should be treated in a special way [van Kleef et al.. 1987]. 3. Identification of covalently bound PQQ

3.1 Introduction

To establish the presence of quinoproteins in mammals, copper-containing amine oxidases (EC 1.4.3.6) seemed very interesting candidates to investigate. Besides copper, these enzymes are known to contain a covalently bound organic cofactor [Yamada and Yasunobu, 1962]. Since the absorption spectra of these enzymes change upon addition of carbonyl-group reagents like hydrazines (see e. g. [Rinaldi et al.. 1983 and Ishizaki and Yasunobu. 1980]). it is generraly accepted that this cofactor has a carbonyl function. This sensitivity for hydrazines is also responsible for the long prevailing conception that PLP is the cofactor. in view of the reactivity of this compound for carbonyl group reagents. It should be realized, however, that PLP as such has never been isolated from a homogeneous copper-containing amine oxidase. On the other hand, these enzymes show a cer­ tain degree of similarity with bacterial methylamine dehydrogenase (MADH) [de Beer et al.. 1980], since they all contain a covalently bound organic cofactor having some characteristics in common. Characterization of the cofactor in MADH (with respect to ESR spectra, redox behaviour and reactivity toward phenylhydra-zines) suggested that it was PQQ or a PQQ like compound [de Beer et al.. 1980 and Kenney and Mclntire. 1983]. However, the organic cofactor in mammalian copper containing amine oxidases is not in its free radical form (as it is in MADH) so that ESR spectroscopy could not be used for identification and anoth­ er approach had to be considered to investigate the possibility of PQQ as a cofactor in these enzymes.

Since free amino acids react with PQQ to a complex mixture of products. detection of this compound in these enzymes by direct hydrolysis was consi­ dered inapplicable (although it has been claimed [Ameyama et al., 1984] that PQQ as such can be detected in these hydrolysates by fluorescense spectroscopy and biological assays). As was recently confirmed, the biological assay fails to detect the amino acid condensation products of PQQ. and fluorescense spectroscopy is not specific enough [van Kleef et al.. 1987 and Adachi et al., 1988]. Therefore, another approach was chosen, the so-called hydrazine method, consisting of the following steps: derivatization of the cofactor with a hydrazine is performed while it is still bound to the enzyme; proteolysis of the derivatized enzyme is carried out with pronase; the cofactor adduct is isolated and compared with m o ­ del compounds prepared from PQQ and the hydrazine used.

The recently developed method by Gallop and coworkers has also been used to analyse covalently bound PQQ [Flueckiger et al.. 1988]. Preparations of diamine

oxidase and dopamine /3-hydroxylase were treated w i t h pronase and PQQ detected by formazan f o r m a t i o n . However, the preparations were impure so t h a t the q u a n ­ t i t a t i v e aspects o f this method are s t i l l unclear.

3.2 The hydrazlne method

Model compounds and products in the enzymes

In the hydrazine method i t is essential that the hydrazine derivative formed is s u f f i c i e n t l y stable towards amino acids, in order to survive the hydrolysis step o f the enzyme. A f t e r extensive screening, i t was found t h a t reaction o f PQQ w i t h 2,4-dinitrophenylhydrazine (DNPH) resulted in the f o r m a t i o n o f such a compound [Lobenstein-Verbeek et al., 1984]. T o elucidate the s t r u c t u r e o f this model c o m ­ pound, an indirect approach was f o l l o w e d . The ethyldimethylester o f PQQ (an intermediate in the chemical synthesis route o f PQQ [Corey and Tramontano, 1981]) reacted w i t h DNPH t o a product yielding crystals suited f o r X - r a y d i f f r a c ­ t i o n analysis. The compound appeared t o be the Z-isomer o f the hydrazone o f the e t h y l d i m e t h y l e s t e r o f PQQ and DNPH at the C(S)-position [van Koningsveld

et al., 19851. H y d r o l y s i s of t h i s hydrazone under m i l d l y alkaline c o n d i t i o n s yielded

a product identical to t h a t prepared f r o m DNPH and PQQ i t s e l f .

Reaction of DNPH w i t h copper-containing amine oxidases (Chapter III) showed t h a t , depending on the conditions during incubation, also another product can be formed which has not been detected so far in the reaction o f PQQ w i t h DNPH. This second product was tentatively suggested to be a tautomeric f o r m , the s o -called azo-compound (Chapter III), since i t has a somewhat higher absorption maximum when compared t o the hydrazone of PQQ and DNPH, suggesting more resonance in the s t r u c t u r e (the differences f o r the t w o products obtained using PH under analogous conditions are even higher, see Chapter I V ) . A l t h o u g h b o t h structures are in f a c t t a u t o m e r s , they do not easily interconvert i n t o each other (only a f t e r prolonged periods o f time at room temperature). Another p r o d u c t , detected in copper-depleted amine oxidases derivatized w i t h DNPH, but not in the model reaction (Chapter II), is probably the hydrazine adduct. A d d i t i o n o f C u2* t r a n s f o r m s this product immediately i n t o the azo-compound. Since i t was found t h a t M A D H was not inhibited by DNPH, while phenylhydrazine (PH) was able to do so (Chapter IV), it was a t t e m p t e d t o obtain the corresponding model c o m ­ pounds of PQQ and PH.

PH tends t o reduce PQQ (like other hydrazines, exept DNPH), which was in fact the reason why the hydrazine method was originally developed w i t h DNPH. However, using conditions developed for reaction o f hydrazines w i t h ketones [Stevens and Higginbotham, 1953], and not more than 10 mg o f PQQ w i t h a s l i g h t molar excess o f PH, t w o products were formed which were similar t o the p r o ­ ducts formed in the enzymes (Chapter IV). Assignment o f the s t r u c t u r e s t o these products (Fig.7) f o l l o w s an analogous reasoning as used f o r the p r o d u c t s o f PQQ and DNPH. A l l products formed w i t h PH as w e l l as w i t h DNPH can be t r a n s ­ formed i n t o PQQ i t s e l f when they are dissolved in dimethylsulphoxide (Chapter IV).

HOOC u COOH "N • HOOC "M HOOC O HOOC u XOOH ,, .COOH r^OH HOOC

hydrazone azo-compound hydrazine adduct

ftm«= « 4 "m> < *ma x = * F i g u r e 7: P o s t u l a t e d a t r u c t u r a a o f P Q Q - D N P H p r o d u c t s .

<*„,a*= *4 5 "m> _{, X m}

_{ax =}

_{° "}

_'

Reaction of the enzyme bound cofactor with hydrazines

The r e s u l t s in Chapters II and IV show that not all hydrazines can be used f o r a particular enzyme. Most probably, the active site o f M A D H (Chapter IV) is unsuited to a l l o w reaction w i t h the bulky DNPH. The same applies to m e t h y l a -mine oxidase (Chapter II) which does not even react w i t h PH (this in analogy w i t h its substrate specific behaviour). The presence o f other c o f a c t o r s may play a crucial role in the reactivity and the type o f product which is f o r m e d . Removal of C u2* f r o m methylamine oxidase (Chapter II) allowed derivatization w i t h DNPH giving a p r o d u c t which is tentatively considered to be the hydrazine adduct o f PQQ and DNPH (Fig.7). Similar phenomena have been reported by others: Suzuki

et al. [1V83] used Cu-depleted, N i - s u b s t i t u t e d bovine serum amine oxidase i n h i ­

bited w i t h PH, and they observed an absorption maximum at 423 nm. Yamada and Yasunobu [ l % 3 ] reported an absorption maximum f o r PH inactivated, C u - d e p l e t e d bovine plasma amine oxidase at 410 nm

In the case o f bovine serum amine oxidase and porcine kidney diamine oxidase (Chapter IV), the conditions applied during derivatization (either w i t h DNPH or PH), determine the product f o r m e d . Hydrazone f o r m a t i o n requires an oxygen atmosphere, and 16 h incubation at 40 ° C . The azo-compound is f o r m e d i m m e ­ diately b u t is only stable in the enzyme under a nitrogen atmosphere (Chapter IV). Dopamine 0-hydroxylase behaved q u i t e differently w i t h PH when compared to the amine oxidases [van der Meer et al., 1988J. A l t h o u g h this enzyme became immediately inhibited after PH addition, no azo-compound was detected while hydrazone f o r m a t i o n required long incubation times. Hydrazone f o r m a t i o n p r o ­ ceeded under atmospheric conditions and even under (semi)-anaerobic conditions. Derivatization o f the copper-depleted enzyme also led t o hydrazone f o r m a t i o n . This example shows t h a t n o t only the conditions during incubation b u t also the nature o f the enzyme determines which product is f o r m e d .

Detachment and purification of the products

Since the products of PQQ and the hydrazine were not stable under s t r o n g l y acid conditions, it was a t t e m p t e d t o detach the products f r o m derivatized enzyme by proteolysis. On testing several proteases, it appeared t h a t sometimes severe losses o f product occurred [Lobenstein-Verbeek et al., 1984 and unpublished r e ­ s u l t s ] , However, application of pronase E detached the p r o d u c t s f r o m the enzymes and resulted in no losses so t h a t this protease m i x t u r e is r o u t i n e l y used

f o r this purpose (see Chapters I I , III and IV), As shown in Chapter I X , t h i s protease preparation can be savely used because it does not contain any PQQ. Perhaps the low y i e l d o f t r y p t i c peptide obtained f r o m porcine kidney diamine oxidase (Chapter V) can also be a t t r i b u t e d to the abovementioned i n s t a b i l i t y o f the PQQ-DNPH hydrazone towards trypsine.

Identification and quantification

As already mentioned, the final step in the hydrazine method consists o f c o m ­ parison o f enzyme-isolated product w i t h the corresponding model compound. Identification is based on spectroscopy ( U V V I S and ' H N M R ) and c h r o m a t o -graphy, methods w e l l accepted for i d e n t i f i c a t i o n of unknown compounds, espe­ cially when used in combination. Nevertheless, doubt has been expressed on the validity of this approach for identification of the c o f a c t o r (see e.g. H a r t m a n and Klinman [1988]). It might, therefore, be reassuring that the products formed f r o m enzymes as w e l l as the model compounds can be t r a n s f o r m e d i n t o PQQ i t s e l f (see above). A f t e r this t r a n s f o r m a t i o n , q u a n t i f i c a t i o n o f PQQ is possible w i t h a biological assay. Also, direct quantification o f the hydrazone formed is possible using its molar absorption coefficient (see Chapters I I I and I V ) .

Evaluation

Derivatization o f an enzyme w i t h a suitable hydrazine can already give an i n ­ dication f o r the presence o f PQQ f r o m the absorption maximum induced. The i n ­ dication is only reliable, however, when excess hydrazine has been removed. This was apparently f o r g o t t e n by T u r and Lerch after derivatizing benzylamine oxidase f r o m Pichia pastoris [ T u r and Lerch, 1988], where the absorption spectrum o f DNPH-treated enzyme shows the absorption band o f DNPH i t s e l f (X = 365 nm, while the hydrazone has i t s absorption maximum at 445 n m , w i t h a S, ,5 o f 31400 M - ' . c m1) .

The conditions applied during derivatization define which product (hydrazone or azo-compound) is f o r m e d . I f this f a c t is not taken i n t o account, errors in i n t e r ­ pretation are inevitable (see f o r example Williamson et al. [1986] where Raman spectroscopy was used to compare the product in the enzyme w i t h the (wrong) model compound). In several recent papers [ W i l l i a m s o n et at., 1986; M o o g et al., 1986 and Knowles et al-, 1986], Raman spectroscopy o f hydrazine-treated amine oxidases was advocated as the t o o l t o provide evidence f o r the existence o f PQQ in amine oxidases. However, although Raman spectra provide a f i n g e r p r i n t o f an unknown compound, i d e n t i f i c a t i o n is problematic when differences occur (and these were in fact found) since these can be ascribed t o either i n t e r a c t i o n of the

derivatized cofactor with local protein structural elements or to a diverging cofactor structure. Moreover, a disadvantage of Raman spectroscopy is that it is not easy to quantify the amount of cofactor in the enzyme. For these reasons. the hydrazine method (as described in Chapter IV with PH) is more reliable in the search for quinoproteins.

!t may be clear from the foregoing that the hydrazine method has been a powerful tool in establishing the quinoprotein nature of several enzymes. How­ ever, it can be imagined that it will fail in certain cases, for instance if the enzyme-bound cofactor is not in the right redox state, or the active site is un-suited to allow reaction of PQQ with the hydrazine. In this respect, a precedent already exists, namely 3,4-dihydroxyphenylalanine (dopa) decarboxylase of pig kid­ ney, where hydrazone formation is limited to only a few percent (Chapter VI). The fact t h a t hydrazone formation with pyridoxal phosphate (PLP) under the conditions applied, is also limited, while PLP-PH hydrazone formation under denaturing conditions [Wada and Snell. 1961] is easily achieved, suggests that the active site does not allow reaction with the hydrazine. Furthermore, it is q u e ­ stionable whether the hydrazine method will be able to determine the total num­ ber of PQQ molecules in an enzyme in all cases. Enzymes ma> show half-of-the-site reactivity, that is that after one PQQ has reacted with the hydrazine, derivatization of the second one may become impossible. This has been used (Chapter 111 > to explain the asymmetry of amine oxidases, i.e. the presence of two Cu2* ions and only one PQQ as detected by the hydrazine method (see e.g.

Chapter III). However, this idea can be rejected in view of the results obtained with the hexanol extraction procedure (Chapter IX).

3.3 The hexanol extraction procedure

Introduction

The drawbacks of the hydrazine method indicated in section 3.2, combined with the attractiveness of an independent second method for the determination and quantification of covalently bound PQQ, and the need for a procedure able to detect PQQ in its products (see section 2.4), resulted in the development of the so-called hexanol extraction procedure. The reasoning behind this procedure is the following: the higher aliphatic alcohol (n-hexanol) has a high boiling point so that refluxing creates the high temperatures required for detachment of PQQ (with HCI). Hexanol forms an adduct with PQQ. which is extracted by the organ­ ic solvent. In this way it escapes undesirable attack by other nucleophiles. The high concentration of strong acid used (3 M HCI) not only detaches PQQ from the protein, but also protonates its carboxylic acid groups so that extraction by the alcohol layer becomes possible. Further reaction in the alcohol layer leads to a stable product, at the same time preventing possible side reactions. Identifica­ tion and quantification of the isolated product is done by comparison with the model compound with respect to its behaviour on a reversed phase h.p.l.c. c o ­ lumn, absorption spectrum, and 'H-NMR parameters.