Binding of NAD+ to rabbit-muscle glyceraldehydephosphate dehydrogenase. The use of N-methylnicotinamidium chloride as a spectral and conformational probe

Biochimica et Biophysica Acta, 328 (1973) 1-9 O Elsevier Scientific P u b l i s h i n g C o m p a n y , A m s t e r d a m - P r i n t e d in T h e N e t h e r l a n d s

BBA

36552

B I N D I N G OF NAD+ TO RABBIT-MUSCLE G L Y C E R A L D E H Y D E P H O S P H A T E DEHYDROGENASE. T H E USE OF N - M E T H Y L N I C O T I N A M I D I U M C H L O R I D E AS A SPECTRAL AND CONFORMATIONAL P R O B E

W. B O E R S a AND J. W. V E R H O E V E N b

aLaboratory of Biochemistry, B.C.P. Jansen Institute, University of Amsterdam, Plantage Muidergracht z2, Amsterdam (The Netherlands) and bLaboratory of Organic Chemistry, University of Amsterdam, Nieuwe Achtergracht z2 9, Amsterdam (The Netherlands) (Received M a y I6th, 1973)

SUMMARY

Addition of N-methylnicotinamidium chloride to rabbit-muscle glyceraldehydephosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD + oxidoreductase (phosphorylating), EC 1.2.1.12) results in the appearance of a charge-transfer absorption spectrum, with an apparent m a x i m u m around 327 nm, that is insensitive to the sulfhydryl reagents iodoacetate and p-chloromercurisulfonate. Resolution of this band and of the Racker band, resulting from the binding of NAD ÷ to the enzyme, gives two overlapping Gaussian absorption bands with maxima at the same wavelength (324 and 369 nm) but with different relative intensities for the two complexes. From these data it is concluded that tryptophan is involved as the electron-donating group in the charge-transfer absorption band of the enzyme-NAD + complex. The different relative intensities of the two overlapping Gaussian absorption bands are attributed to dissimilarities in the relative orientation of the nicotinamidium ion of NAD + and N-methylnicotinamidimn chloride, respectively, with respect to the tryptophan molecule involved. By using the charge-transfer absorption band of the enzyme-N-methylnicotinamidium complex as a conformational probe, it is shown that the effect on the protein conformation, caused by the binding of the first two NAD + molecules to the enzyme, is different from that brought about b y the binding of the third and fourth NAD+ molecule.

INTRODUCTION

Binding of NAD + to glyceraldehydephosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD + oxidoreductase (phosphorylating), EC 1.2.1.12) results in the appearance of a broad absorption band with an apparent m a x i m u m near 360 nm 1.

2

w . BOERS, J. W. VERHOEVEN

There have been several attempts to interpret this absorption spectrum. Racker and Krimsky 1 suggested the presence of a quinoid-type structure in the nicotinamide moiety of NAD +, and since the absorption band disappeared on the addition of sulfhydryl-reacting agents such as iodoacetic acid, it was assumed that the sulfhydryl group of a cysteine residue in the enzyme might form a covalent bond with some site on the nicotinamidium ring of the coenzyme. Kosower ~ supposed that this broad, diffuse absorption band might be assigned to a charge-transfer interaction 3 between the enzyme and the coenzylne, in analogy to the occurrence of charge-transfer complexes formed from I-methylpyridinium ions and electron donors. Using model compounds, Cilento and coworkers 4,5 and Shifrin 6,7 have shown indeed that a charge-transfer complex arises as a result of the interaction of an efficient electron donor, such as the indole side chain of tryptophan, with a moderately efficient electron acceptor, such as the pyridinium ring of NAD +. The absorption maximum, the extinction coefficient and the shape of the corresponding charge-transfer transition are similar to those of the Racker band 1 of the NAD+-glyceraldehydephosphate dehydrogenase complex. Polgar s, on the other hand, assumed that a nucleophilic thiol group of the enzyme, in the mercaptide ion form, serves as the electron donor. Van Eys and Kaplan 9 have shown that various mercaptide ions and pyridinium compounds can form complexes with absorption maxima around 33o-34 ° n m . These observations were confirmed by Boross and Cseke ~° who concluded from the position of the absorption m a x i m u m and the similarity of the pH dependence of the absorption spectra of the enzyme-NAD + complex and the mercaptide ion-pyridinium complex (the tryptophan NAD + charge-transfer spectrum shows the opposite pH dependence), that the active thiol group n of glyceraldehydephosphate dehydrogenase is directly involved in the chromophoric part of the enzyme-coenzyme complex. In this paper we bring forward further experiments in an a t t e m p t to establish the electron-donating group in the charge-transfer complex between NAD+ and the enzyme. Our attention was drawn to this problem when we noticed that upon addition of NAD + to the enzyme the absorbance at 360 nm still increases 12 after the four specific NAD + molecules is are bound. Two explanations of this phenomenon are possible: (I) it is due to a charge-transfer complex formed between NAD+ and an electron-donating group in a binding site for NAD + with very low affinity; (2) it is due to 'contact' charge-transfer of the type described by Orgel and Mulliken 14. As a model for the pyridinium ring of the NAD ~ molecule, the part of this molecule that could be involved in a charge-transfer complex, we use N-methylnicotinamidium chloride, first introduced by Karrer et al. 15. As shown by Deranlau et al. 16-1s with lysozyme, this molecule can identify tryptophan molecules partially or completely exposed to the solvent surrounding the protein, by giving a chargetransfer absorption band. This absorption band can function as a conformational probe in proteins when other molecules that also react with the protein are added. This paper describes also what happens to the charge-transfer band, resulting from the interaction of N-methylnicotinamidium chloride and glyceraldehydephosphate dehydrogenase, when the four specific NAD + molecules TM are successively bound to the enzyme.

GLYCERALDEHYDEPHOSPHATE DEHYDROGENASE

3

N-Methylnicotinamidium chloride as a model for N A D + in the identification of the electron-donating group of the charge-transfer absorption band N-Methylnicotinamidium chloride can interact with glyceraldehydephosphate dehydrogenase to form a charge-tranfer complex. The absorption spectrum resulting from this interaction, measured by means of difference spectroscopy, is shown in Fig. IA. This spectrum is identical with that obtained from charge-transfer interaction of N-methylnicotinamidium with tryptophan model compounds 1~ or with a tryptophan moiety in lysozyme16, is and can, therefore, be ascribed to interaction of N-methylnicotinamidium with one or more tryptophan moieties in the enzyme. For

~A

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.. -~5~:-... .010

33

30

25

20

'~'/,,/(c rdt x10-3) Fig. IA. C h a r g e - t r a n s f e r a b s o r p t i o n (-) a n d t h e t w o - b a n d G a u s s i a n a p p r o x i m a t i o n (. . . . . ), i n d u c e d b y a d d i t i o n of N - m e t h y l n i c o t i n a m i d i u m chloride to c h a r c o a l - t r e a t e d g l y c e r a l d e h y d e p h o s p h a t e d e h y d r o g e n a s e , m e a s u r e d as t h e a b s o r p t i o n difference b e t w e e n t h e e n z y m e (36.5/xM) to w h i c h N - m e t h y l n i c o t i n a m i d i u m chloride (IOO raM) is a d d e d a n d t h e s e p a r a t e solutions of t h e s e c o m p o n e n t s , dissolved in ioo m M TIis-HC1 (pH 8.2) buffer, c o n t a i n i n g 5 m M E D T A , 2o °C. Effective light p a t h , 2 cm.

~A

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3

30

25

20

~/(crn-lx10-3)

Fig. I B. T h e R a c k e r b a n d ( - - ) of g l y c e r a l d e h y d e p h o s p h a t e d e h y d r o g e n a s e a n d t h e t w o - b a n d G a u s s i a n a p p r o x i m a t i o n (. . . . . ), m e a s u r e d as t h e a b s o r p t i o n difference b e t w e e n t h e e n z y m e (60 /~M) to w h i c h four N A D + molecules are b o u n d a n d t h e s e p a r a t e solutions o f t h e s e c o m p o n e n t s , dissolved in i o o m M Tris-HC1 (pH 8.2) buffer, c o n t a i n i n g 5 m M E D T A ; 20 °C. Effective light p a t h , i cm.

4

W. BOERS, J. W. VERHOEVEN

comparison, the s p e c t r u m of the e n z y m e - N A D + complex, the so-called R a c k e r b a n d ~, when four N A D + molecules are b o u n d to the enzyme, is p l o t t e d in Fig. I B . The s p e c t r a differ in shape a n d in the position of the a p p a r e n t a b s o r p t i o n m a x i m a , viz. 30 5oo cm -1 (327 nm) a n d 28 2oo cm -1 (355 nm) for N - m e t h y l n i c o t i n a m i d i u m a n d N A D ÷, respectively. Moreover, a d d i t i o n of s u l f h y d r y l reagents such as i o d o a c e t a t e or p - c h l o r o m e r c u r i s u l f o n a t e abolish the b a n d with N A D + b u t have no effect at all on t h a t w i t h N - m e t h y l n i c o t i n a m i d i u m . I n a previous p a p e r ~2 we have shown t h a t after the four specific NAD+ molecules 13 are b o u n d to c h a r c o a l - t r e a t e d g l y c e r a l d e h y d e p h o s p h a t e dehydrogenase, the a b s o r p t i o n at 36o n m still increases when more N A D + is added. The a b s o r p t i o n spect r u m , also m e a s u r e d b y difference spectroscopy, of this aspecific complex shown in Fig. 2 is similar to t h a t of the e n z y m e - N - m e t h y l n i c o t i n a m i d i u m complex. I t t h u s seems reasonable to a t t r i b u t e the a b s o r p t i o n due to this 'fifth' NAD+ molecule, to i n t e r a c t i o n w i t h t r y p t o p h a n moieties of the enzyme. Z~A

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'~'~/(cm-1 x10-3) Fig. 2. Charge-transfer absorption ( - - ) and the two-band Gaussian approximation (..... ) induced by the binding of the" fifth" NAD + molecule to charcoal-treated glyceraldehydephosphate dehydrogenase, measured as the absorption difference between the enzyme (4o.5 #M) to which four specific NAD+ molecules are bound and this enzyme-NAD + complex to which 1.3 mM NAD + is added and the separate solutions of the components, dissolved in ioo mM Tris-HCl buffer (pH 8.2), containing 5 inM EDTA; 20 °C. Effective light path, 2 cm. The s p e c t r a shown in Figs I A a n d I B can be resolved into two overlapping Gaussian a b s o r p t i o n b a n d s with m a x i m a at t h e same w a v e l e n g t h (324 a n d 369 nm) b u t with different r e l a t i v e intensities for the two c o m p o n e n t s (see Table I). I t is not u n u s u a l t h a t c h a r g e - t r a n s f e r a b s o r p t i o n b a n d s with p y r i d i n i u m ions are composed of two bands. This was a t t r i b u t e d b y Kosower et al. 19 to two excited states of the eleet r o n - d o n a t i n g group, b u t Verhoeven et al. 2° h a v e suggested t h a t t h e two b a n d s could be ascribed to the presence of two v a c a n t molecular orbitals in the p y r i d i n i u m ion, since t h e y could show t h a t the energy of the two a b s o r p t i o n b a n d s w i t h the cyanos u b s t i t u t e d d e r i v a t i v e s of N - m e t h y l p y r i d i n i u m iodide is d e t e r m i n e d b y the position of the c y a n o group on t h e p y r i d i n i u m ion. Their e x p l a n a t i o n has been r e c e n t l y confirmed b y Kosower et al. ~1 a n d M a c K a y a n d P o z i o m e k 22. Verhoeven et al. 2° calculated t h a t for t h e N - m e t h y l - 3 - c y a n o p y r i d i n i u m ion these two b a n d s m u s t be 0.47 eV a p a r t . The two Gaussian a b s o r p t i o n b a n d s of the Figs I A a n d I B , which also described

GLYCERALDEHYDEPHOSPHATEDEHYDROGENASE

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TABLE I RELATIVE

AREAS

AND

BAND

MAXIMA

MUSCLE GLYCERALDEHYDEPHOSPHAT~

FOR

CHARGE--TRANSFER

DEHYDROGENASE

WITH

ABSORPTION DIFFERENT

BANDS

OF

ACCEPTORS:

RABBITGAUSSIAN

APPROXIMATION

A cceptor

Four specific NAB +1~ "Fifth" NAD +1~ N-Methylnicotinamidium

Short-wavelength band

Long-wavelength band

Smaz (rim)

Area (%)

~max (nm)

Area (%J

324 322 324

45 7° 65

369 360 369

55 3° 35

complexes with 3-substituted pyridinium ions, differ 3700 cm -1 or 0.46 eV, in excellent agreement with the energy differences found by Verhoeven et al. 2° and by Deranleau et al. l" for the resolved bands given by the lysozyme-N-methylpyridinium complex. Thus the same electron-donating group or at least groups with the same ionization potential must be involved in the complexes with the enzyme. Since in the complex with N - m e t h y l n i c o t i n a m i d i u m the electron-donating group is very likely tryptophan, it is reasonable to assume that this molecule is also involved as the electron-donating group in the Racker band 1 of glyceraldehydephosphate dehydrogenase. This leaves the difference in relative intensities and the different behavior towards sulfiaydryl reagents still to be explained. Two explanations are possible for the fact that sulfhydryl reagents, such as iodoacetate and p-chloromercurisulfonate abolish the Racker band : (I) the binding of these reagents induces a conformational change in the enzyme so that a charge-transfer interaction between the nicotinamidium group and a tryptophan molecule is impossible, (2) the cysteine molecule 'in the active centre' is situated in the neighbourhood of the tryptophan molecule that is involved in the charge-transfer complex, and binding of iodoacetate or :#-chloromercurisulfonate to this cysteine molecule abolishes the charge-transfer complex by steric hindrance. We favour the second explanation. Boross and CsekO ° and Boross 2~showed that native glyceraldehydephosphate dehydrogenase from swine muscle is able to form a ternary Ag+-enzyme-NAD + complex with Ag +, in which Ag+ is bound by the active thiol groups of the enzyme. The Racker band does not disappear by this treatment but its shape is changed, and the absorption m a x i m u m is shifted from 355 to 335 nm. The absorption spectrum of this ternary complex is, in fact, similar to that of the enzyme-N-methylnicotinamidium complex, and can be resolved into two overlapping Gaussian absorption bands with maxima at approx. 325 and 365 nm, with relative intensities similar to those of the enzyme-N-methylnicotinamidium complex. Thus when a relatively small ion is bound to the active thiol group, a charge-transfer complex of NAD÷ with tryptophan is still possible. The insensitivity of the enzymeN - m e t h y l n i c o t i n a m i d i u m complex towards sulfllydryl reagents m a y be due either to the fact that N-methylnicotinamidium is less sensitive to steric hindrance than NAD+ or that N-methylnicotinamidium does not (exclusively) interact with the same tryptophan molecules as the first four NAD+ molecules. As shown above the Racker band 1 and the absorption band of the enzymeN-methylnicotinamidium complex are composed of the same two charge-transfer bands as the NAD+ complex, but with different relative intensities (see Table I).

6

W. BOERS, J. w . VERHOEVEN

This could indicate that the relative orientation of the electron acceptor (i.e. the nicotinamidium ion) and the electron donor (i.e. tryptophan) is different for these complexes. It has been shown both theoretically 24 and experimentally 2s that the intensity of charge-transfer bands depends upon the overlap of the molecular orbitals involved and thereby upon the relative orientation of donor and acceptor. Since, as stated above, different acceptor orbitals are involved in the 'resolved' charge-transfer transitions at 324 and 36 9 nm, the relative intensity of these transitions can be expected to be conformation dependent. The interaction of the adenine moiety of NAD + with its corresponding subsite provides the major contribution to the energy of binding to the enzyme 26. The specific binding of the adenine moiety will restrict the movement of the nicotinamidium group of NAD + so that it cannot freely interact with a tryptophan molecule as is the case for the N-methylnicotinamidium ion. The fact that after modifying the cysteine molecule with Ag + the spectrum of the Racker band becomes similar to that of the enzyme-N-methylnicotinamidium complex is perhaps an indication that the active cysteine molecule is involved in the orientation of the ring system of the nicotinamidium group in such a way that it interacts with a tryptophan molecule, and as a consequence the nicotinamidium group as well as the cysteine molecule m a y be in a transition state that promotes efficient catalysis, indirectly caused by a tryptophan molecule.

The use of the charge-transfer band of the enzyme-N-methylnicotinamidium complex as a conforma~ional probe The charge-transfer absorption band resulting from the interaction of Nmethylnicotinamidium chloride with enzyme tryptophans can be used as a conformational probe for the enzyme, as has been demonstrated for lysozyme 1G-is. NMethylnicotinamidium reveals tryptophan molecules that are partially or completely exposed to the solvent surrounding the protein, although the hydrophobic character of tryptophan suggests that it should be located predominantly in the interior of globular proteins 27. Of the four potential indole ring binding sites for N-methylnicotamidium Trp-62 appears to be more or less completely available for a m a x i m u m overlap 2s. Due to the low association constant of N-methylnicotinamidium with glyceraldehydephosphate dehydrogenase and the instability of the enzyme in the presence of high concentrations of N-methylnicotinamidium chloride, it was not possible directly to determine the dissociation constant of the complex. However, using the absorption coefficient found for the lysozyme-N-methylnicotinamidium complex 16, lO4O M-1.cm 1 at 35 ° n m , we can calculate from the spectrum in Fig. IA that the dissociation constant is 32o mM, in good agreement with the one found for lysozyme 16 (31o mM). This could mean that only one tryptophan molecule in the apoenzyme is available for complex formation with N-methylnicotinamidium chloride, but the structure of the enzyme, a tetramer composed of four identical subunits 29, makes it more likely that two a° or four tryptophan molecules are available with correspondingly lower association constants, and that the calculated binding constant is only apparent. The effect of addition of NAD + to charcoal-treated glyceraldehydephosphate dehydrogenase on the charge-transfer absorption band of the interaction of Nmethylnicotinamidium with the enzyme is shown in Fig. 3. The first two NAD +

7

GLYCERALDEHYDEPHOSPHATE DEHYDROGENASE 040

AA

,030

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.- .

\

.......... . . .

\

\

l ..

-,. •

..

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Fig. 3. Effect of binding of N A D + on the charge t r a n s f e r a b s o r p t i o n arising from the interaction of N - m e t h y l n i c o t i n a m i d i u m chloride with charcoal-treated glyceraldehydephosphate dehydrogenase (@ Fig. IA), m e a s u r e d as the a b s o r p t i o n difference between e n z y m e - N A D + complexes (37/zM) of v a r y i n g compositions to which N - m e t h y l n i c o t i n a m i d i u m chloride (ioo raM) is added and the separate solutions of these two components. - - - - - - , o.t or 2 N A D + molecules b o u n d per molecule enzyme; . . . . . , 3 NAD+ molecules b o u n d ; ..... , 3.65 N A D e molecules bound. Buffer ioo mM Tris-HC1 (pH 8.2), containing 5 mM E D T A ; 2o °C. Effective light path, 2 cm.

molecules bound to the enzyme have no effect on the charge-transfer band, which means that (i) NAD + binds to other tryptophan molecules and (ii) the first two NAD + molecules bound to the enzyme do not induce a conformational change that can be detected by the charge transfer band of the tryptophan molecule that is exposed to the solvent surrounding the protein. Binding of the third and fourth NAD+ molecule increases the intensity of the absorption band. Since absorption coefficients measured with model compounds of the indole-pyridinium type are all of the order of 100017, it is unlikely that this increased intensity is due to an increase in the absorption coefficient. It is more likely that as a result of conformational changes in the protein, tryptophan molecules become more exposed to the solvent. Rossmann et al. ~1 and Gorjunov et al. 32 have demonstrated by X-ray studies that holo-glyceraldehydephosphate dehydrogenase from lobster and human muscle, respectively, has three intersecting 2-fold axes of symmetry. Gorjunov et al. 32 showed further that there was a transformation of the molecular s y m m e t r y of the enzyme during the binding of NAD÷, which they interpreted as a c h a n g e from a 222molecular s y m m e t r y in the holoenzyme to a s y m m e t r y lower than 222 in the apoenzyme. In view of this it is possible that in the apoenzyme only one tryptophan molecule is available for charge-transfer interaction with N-methylnicotinamidium chloride, whereas in the holoenzyme two tryptophan molecules are available, in accordance with the observed molecular symmetry. EXPERIMENTAL

Glyceraldehydephosphate dehydrogenase was isolated from rabbit muscle by the method of Cori et al. z3, slightly modified by Hilvers 34. The specific activity, measured spectrophotometrically using DL-glyceraldehyde 3-phosphate, NAD + and phosphate by the method of Ferdinand sS, was 145-165/zmoles NADH/min per mg

8

w . BOERS, J. w , VERHOEVEN

protein. (The correction factor of 1.0735 for n o n - s a t u r a t i o n with respect to p h o s p h a t e was n o t used). NAD+ was r e m o v e d b y stirring with charcoal (0.2 g/ml solution; cf. ref. 36). The enzyme concentration was calculated from absorbance m e a s u r e m e n t s a t 280 nm, using the e x t i n c t i o n coefficients r e p o r t e d b y F o x a n d D a n d l i k e r 37. A molecular weight of 145 000 was assumed 29. N - M e t h y l n i c o t i n a m i d i u m chloride was p r e p a r e d from the corresponding iodide b y r e p e a t e d equilibration with an excess of AgC115, or was o b t a i n e d as a commercial p r e p a r a t i o n (Sigma). I o d o a c e t i c acid (The British D r u g Houses Ltd) was recrystallized from carbon t e t r a c h l o r i d e and dried before use. All other m a t e r i a l s were o b t a i n e d from c o m m e r c i a l sources a n d were used w i t h o u t purification. A b s o r b a n c e m e a s u r e m e n t s were carried out with a Cary recording spectrophotometer, Model I7, fitted w i t h a t e m p e r a t u r e - r e g u l a t e d cell holder. Difference s p e c t r a measured, at 20 °C, in t a n d e m double cells essentially according to the procedure of H e r s k o v i t s a n d L a s k o w s k i 38, b u t modified b y S w a n e y 39, since the c h a r g e - t r a n s f e r a b s o r p t i o n b a n d s of the e n z y m e - N A D + complex and the e n z y m e ( - N A D + ) - N m e t h y l n i c o t i n a m i d i u m complex are s i t u a t e d in the same spectroscopic region. B y using this modified technique the errors m a d e b y p i p e t t i n g the solutions are minimized. Slit w i d t h a n d d y n o d e voltage were chosen to give m a x i m a l l y resolved s p e c t r a h a v i n g a m i n i m u m of noise b a c k g r o u n d . U n d e r these conditions Beer's law was followed. The Gaussian a p p r o x i m a t i o n s of the s p e c t r a were m a d e on a D u P o n t m o d e l 310 curve resolver. ACKNOWLEDGEMENTS W e wish to t h a n k Professor E. C. Slater for s t i m u l a t i n g discussions a n d v a l u a b l e criticism a n d Miss G. J. M. de Bruin for skilful technical assistance. This work was s u p p o r t e d in p a r t b y g r a n t s from the N e t h e r l a n d s O r g a n i z a t i o n for the A d v a n c e m e n t of Pure Research (Z.W.O.) under the auspices of the N e t h e r l a n d s F o u n d a t i o n for Chemical Research (S.O.N.). REFERENCES I 2 3 4 5 6 7 8 9 io ii 12 13 14 15 16

Racker, E. and Krimsky, I. (1952) J. Biol. Chem. 198, 731-743 Kosower, E. M. (1956) J. A m . Chem. Soc. 78, 3497-35Ol Mulliken, R. S. (1952) J. A m . Chem. Soc. 74, 811-824 Cilento, G. and Giusti, P. (1959) J. A m . Chem. Soc. 81, 38Ol-38o2 Cilento, G. and Tedeschi, P. (1961) J. Biol. Chem. 236, 9o7-91o Shifrin, S. (1964) Biochim. Biophys. Acta 81, 2o5-213 Shifrin, S. (1969) A n n . N . Y . Acad. Sci. 158, 148-16o Polg~r, L. (1964) Experientia 20, 4o8-413 Van Eys, J. and t(aplan, N. O. (1957) J. Biol. Chem. 228, 3o5-314 Boross, L. and Cseke, E. (1967) Acta Biochim. Biophys. Acad. Sci. Hung. 2, 47-57 Harris, I., Meriwether, 13. P. and Park, J. H. (1963) Nature 198, 154-157 Boers, W., Oosthuizen, C. and Slater, E. C. (1971) Biochim. Biophys. Acta 25o, 35-46 De Vijlder, J. J. M. and Slater, E. C. (1968) Biochim. Biophys. Acta 167, 23-34 Orgel, L. E. and Mulliken, R. S. (1957) J. A m . Chem. Soc. 79, 4839-4846 Karrer, P., Schwarzenbach, G., Benz, F. and Solmssen, U. (1936) Helv. Chim. Aeta 19, 811-828 Deranleau, D. A., Bradshaw, R. A. and Schwyzer, R. (1969) Proc. Natl. Acad. Sci. U.S. 63,885889 17 Deranleau, D. A. and Schwyzer, R. (197o) Biochemistry 9, 126-134 18 Bradshaw, R. A. and Deranleau, D. A. (197o) Biochemistry 9, 331o-3315

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19 Kosower, E. M., Skorcz, J. A., Schwarz, Jr, W. M. and Patton, J. W. (196o) J. Am. Chem. Soc. 82, 2188-2191 20 Verhoeven, J. W., Dirkx, I. P. and De Boer, Th. J. (1969) Tetrahedron 25, 3395-3405 21 Kosower, E. M., Land, E. J. and Swallow, A. J. (1972) J. Am. Chem. Soc. 94, 986-987 22 MacKay, R. A. and Poziomek, E. J. (1972) J. Am. Chem. Soe. 94, 4167-417 ° 23 Boross, L. (1965) Biochim. Biophys. Acta 96, 52-60 24 Anderson, G. R. (197 o) J. Am. Chem. Soc. 92, 3552-3560 25 Schroff, L. G., Van der Weerdt, A. J. A., Staalman, D. J. H., Verhoeven, J. W. and De Boer, Th. J. (1973) Tetrahedron Letters, 1649-1652 26 Yang, S. T. and Deal, Jr, W. C. (1969) Biochemistry 8, 28o6-2813 27 Tanford, C. (1964) J. Am. Chem. Soc. 86, 2050-2059 28 Blake, C. C. F., Johnson, L. N., Mair, G. A., North, A. C. T., Phillips, D. C. and Sarma, V. R. (1967) Proc. R. Soc. London, Ser. B, 167, 378-388 29 Harris, J. I. and Perham, R. N. (1965) J. Mol. Biol. 13, 876-884 3° Malhotra, O. P. and Bernhard, S. A. (1968) J. Biol. Chem. 243, 1243-1252 31 Rossmann, M. G., Ford, G. C., Watson, H. C. and Banaszak, L. J. (1972) J. Mol. Biol. 64, 237 249 32 Gorjunov, A. I., Andreeva, N. S., Baranowski, T. and Wolny, M. (1972) J. Mol. Biol. 69, 421 426 33 Cori, G. T., Slein, M. W. and Cori, C. F. (1948) J. Biol. Chem. 173, 6o5-618 34 Hilvers, A. G. (1964) Ph.D. thesis, University of Amsterdam, pp. 23-28, Hofman, Alkmaar 35 Ferdinand, W. (1964) Biochem. J. 92, 578-585 36 Velick, S. F., Hayes, Jr, J. E. and Harting, J. (1953) J. Biol. Chem. 203, 527 544 37 Fox, Jr, J. B. and Dandliker, W. B. (1956) J. Biol. Chem. 221, lOO5-1Ol 7 38 Herskovits, T. T. and Laskowski, Jr, M. (1962) J. Biol. Chem. 237, 2481-2492 39 Swaney, J. B. (1971) Anal. Biochem. 43, 388-393