The mechanism of succinate or fumarate transfer in the tricarboxylic acid cycle allows molecular rotation of the intermediate.

Mitochondria were incubated with L[5-13C]glutamic acid and the distribution of the label between the two carboxyl carbon atoms of the L-aspartic acid formed was determined by 13C NMR. The reaction sequence leading from L-glutamic acid to L-aspartic acid spans the tricarboxylic acid cycle reactions involving the two symmetrical intermediates succinate and fumarate. The C2 symmetry of these intermediates in principle permits a discrimination of the mechanism of their transfer between their enzyme sites of production and utilization. A direct transfer of metabolite from site to site by translation alone predicts an unequal distribution of 13C between the C1 and C4 of aspartate, whereas molecular rotation during transfer allows for a scrambling of the original C5 label. Under several conditions of different glutamate concentrations and solvent osmotic pressures, equal labeling in the C1 and C4 carbons of aspartate is observed. This observation is inconsistent with a transfer mechanism restricting molecular rotation for both intermediates but is compatible with both a random diffusion and a direct transfer mechanism provided the latter allows molecular rotation.

Direct transfer of NADH between alpha-glycerol phosphate dehydrogenase and lactate dehydrogenase: fact or misinterpretation?

Following the criticism by Chock and Gutfreund [Chock, P.B. & Gutfreund, H. (1988) Proc. Natl. Acad. Sci. USA 85, 8870-8874], that our proposal of direct transfer of NADH between glycerol-3-phosphate dehydrogenase (alpha-glycerol phosphate dehydrogenase, alpha-GDH; EC 1.1.1.8) and L-lactate dehydrogenase (LDH; EC 1.1.1.27) was based on a misinterpretation of the kinetic data, we have reinvestigated the transfer mechanism between this enzyme pair. By using the "enzyme buffering" steady-state kinetic technique [Srivastava, D.K. & Bernhard, S.A. (1984) Biochemistry 23, 4538-4545], we examined the mechanism (random diffusion vs. direct transfer) of transfer of NADH between rabbit muscle alpha-GDH and pig heart LDH. The steady-state data reveal that the LDH-NADH complex and the alpha-GDH-NADH complex can serve as substrate for the alpha-GDH-catalyzed reaction and the LDH-catalyzed reaction, respectively. This is consistent with the direct-transfer mechanism and inconsistent with a mechanism in which free NADH is the only competent substrate for either enzyme-catalyzed reaction. The discrepancy between this conclusion and that of Chock and Gutfreund comes from (i) their incorrect measurement of the Km for NADH in the alpha-GDH-catalyzed reaction, (ii) inadequate design and range of the steady-state kinetic experiments, and (iii) their qualitative assessment of the prediction of the direct-transfer mechanism. Our transient kinetic measurements for the transfer of NADH from alpha-GDH to LDH and from LDH to alpha-GDH show that both are slower than predicted on the basis of free equilibration of NADH through the aqueous environment. The decrease in the rate of equilibration of NADH between alpha-GDH and LDH provides no support for the random-diffusion mechanism; rather, it suggests a direct interaction between enzymes that modulates the transfer rate of NADH. Thus, contrary to Chock and Gutfreund's conclusion, all our experimental data compel us to propose, once again, that NADH is transferred directly between the sites of alpha-GDH and LDH.

Noncovalent modulation by ATP of the acyl transfer from acyl-glyceraldehyde-3-phosphate dehydrogenase to phosphate.

The effect of ATP on the formation, spectral properties, and reactions of [beta-(2-furyl)acryloyl]glyceraldehyde-3-phosphate dehydrogenase (FA-GPDH) has been investigated. The chromophoric FA-GPDH has the advantage of providing spectrophotometric signals of the interaction of acyl enzyme with nucleotides and dinucleotides. The results are consistent with the exclusive existence of two acyl-enzyme conformations previously inferred from the interaction of the acyl enzyme with NAD+ and NADH. ATP interaction stabilizes a conformation different from that stabilized by NAD+. The inhibitory effects of ATP on these reactions are consistent with the reported inhibitory effect of ATP on the steady-state reaction with the true substrate. The physiological significance of these results to the regulation of glycolysis, via the ligand-dependent fate of 3-phosphoglycerol-GPDH, is discussed.

The intracellular equilibrium thermodynamic and steady-state concentrations of metabolites.

A new model for the organization and flow of metabolites through a metabolic pathway is presented. The model is based on four major findings. (1) The intracellular concentrations of enzyme sites exceed the concentrations of intermediary metabolites that bind specifically to these sites. (2) The concentration of the excessive enzyme sites in the cell is sufficiently high so that nearly all the cellular intermediary metabolites are enzyme-bound. (3) Enzyme conformations are perturbed by the interactions with substrates and products; the conformations of enzyme-substrate and enzyme-product complexes are different. (4) Two enzymes, catalyzing reactions that are sequential in a metabolic pathway, transfer the common metabolite back and forth via an enzyme-enzyme complex without the intervention of the solvent environment. The model proposes that the enzyme-enzyme recognition is ligand-induced. Conversion of E2S and E2P results in the loss of recognition of E2 by E1 and the concomitant recognition of E2 by E3. This model substantially alters existent views of the bioenergetics and the kinetics of intracellular metabolism. The rates of direct transfer of metabolite from enzyme to enzyme are comparable to the rates of interconversion between substrate and product within an individual enzyme. Consequently, intermediary metabolites are nearly equipartitioned among their high-affinity enzyme sites within a metabolic pathway. Metabolic flux involves the direct transfer of metabolite from enzyme to enzyme via a set of low and nearly equal energy barriers.

Mechanism of transfer of reduced nicotinamide adenine dinucleotide among dehydrogenases. Transfer rates and equilibria with enzyme-enzyme complexes.

The direct transfer of NADH between A-B pairs of dehydrogenases and also the dissociation of NADH from individual E-NADH complexes have been investigated by transient stopped-flow kinetic techniques. Such A-B transfers of NADH occur without the intermediate dissociation of coenzyme into the aqueous solvent environment [Srivastava, D.K., & Bernhard, S.A. (1985) Biochemistry 24, 623-628]. The equilibrium distributions of limiting NADH among aqueous solvent and A and B dehydrogenase sites have also been determined. At sufficiently high but realizable concentrations of dehydrogenases, both the transfer rate and the equilibrium distribution of bound NADH are virtually independent of the excessive enzyme concentrations; at excessive E2 concentration, substantial NADH is bound to the E1 site. These results further substantiate earlier kinetic arguments for the preferential formation of an EA-NADH-EB complex, within which coenzyme is directly transferred between sites. The unimolecular specific rates of coenzyme transfer from site to site are nearly invariant among different A-B dehydrogenase pairs. The equilibrium constants for the distribution of coenzyme within the EA X EB complexes are near unity. At high [E2] and for [E2] greater than [E1] greater than [NADH], E1-NADH X E2 and E1 X NADH-E2 are virtually the only coenzyme-contained species. In contrast to the nearly invariant unimolecular NADH transfer rates within EA X EB complexes, unimolecular specific rates of dissociation of NADH from E-NADH into aqueous solution are highly variable.(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolite transfer via enzyme-enzyme complexes.

The concentrations of enzyme sites in cells are usually higher than the concentrations of cognate intermediary metabolites. Therefore metabolic pathways or substantial segments of pathways may proceed by the direct transfer of metabolites from one enzyme site to the next by means of enzyme-enzyme complex formation. This mechanism of metabolite transfer differs from that usually assumed where dissociation and random diffusion of metabolite through the aqueous environment is responsible for the transfer to the next enzyme site. Since the direct transfer mechanism does not involve the aqueous environment, the energetics of metabolite interconversion can differ from expectations based on aqueous solution data. Evidence is summarized suggesting that metabolite is transformed and transferred with equal facility everywhere in the direct transfer pathway.

Chromophoric cinnamic acid substrates as resonance Raman probes of the active site environment in native and unfolded alpha-chymotrypsin.

Chromophoric [4-(dimethylamino)cinnamoyl]imidazole reacts with the serine protease alpha-chymotrypsin to form an acyl enzyme. At pHs below 4.0, the acyl enzyme turns over very slowly to yield the free acid. During this slow deacylation it is possible to obtain a very good resonance Raman spectrum of the acyl intermediate by using the 350.7-nm line of the krypton laser. The resonance Raman carbonyl frequency of the covalently bonded substrate and its wavelength at maximum intensity in the absorption spectrum of the acyl enzyme have been taken and used to monitor the active site environment. A comparison has been made of the absorption and Raman spectra of the acyl enzyme and those of the corresponding chromophoric methyl ester, aldehyde, and imidazole model compounds. A linear correlation is found between the wavelength of maximum absorption and the Raman frequency of the carbonyl group over a wide range of solvent conditions for each of the model compounds. By combining the Raman carbonyl frequency with the absorption maximum, we can determine that the bond order changes in the carbonyl bond of the bound substrate are not due to changes in the solvent, since the carbonyl frequency and the absorption maximum of the acyl enzyme do not fall on any of the linear correlations for the model compounds. The unusual spectroscopic properties of the bound substrate appear to be due to some specific enzyme-induced change in the substrate when it is bound at the active site. Thermal unfolding of the acyl enzymes changes both the carbonyl frequency of the acyl enzyme and its absorption maximum to completely different values.(ABSTRACT TRUNCATED AT 250 WORDS)

Multiple ion-dependent and substrate-dependent Na+/K+-ATPase conformational states. Transient and steady-state kinetic studies.

The hydrolysis of beta-(2-furyl)acryloyl phosphate (FAP), catalyzed by the Na+/K+-ATPase, is faster than the catalyzed hydrolysis of ATP. This is due to catalyzed hydrolysis of the pseudosubstrate by K+-dependent states of the enzyme, thus bypassing the Na+-dependent enzyme states that are required and are rate limiting in ATP hydrolysis. Unlike ATP, FAP is a positive effector of the E2 state. A study of FAP hydrolysis permits a detailed analysis of later steps in the overall ion translocation-ATP hydrolysis pathway. During the steady state of FAP hydrolysis in the presence of K+, substantial phosphoryl-enzyme is formed, as is indicated by the covalent incorporation of 32P from [32P]FAP. A comparison of the phosphoryl-enzyme yield with the rate of overall hydrolysis reveals that at 25 degrees C the phosphoryl-enzyme formed is all kinetically competent. Both the yield of phosphoryl-enzyme and the rate of overall hydrolysis of FAP are [K+] dependent. The transition E1 in equilibrium E2 is also [K+] dependent, but the rate of transition is differently affected by [K+] than are the above-mentioned two processes. Two distinct roles for K+ are indicated, as an effector of the E1-E2 equilibrium and as a "catalyst" in the hydrolysis of the E2-P. In contrast to the results at 25 degrees C, a virtually stoichiometric yield of phosphoryl-enzyme occurs at 0 degree C in the presence of Na+ and the absence of K+. At lower concentrations of K+ and in the presence of Na+, the hydrolysis of FAP at 0 degree C proceeds substantially through the E1-E2 pathway characteristic of ATP hydrolysis. The selectivity of FAP for the E2-K+-dependent pathway is due to the thermal inactivation of E1 at 25 degrees C in the absence of ATP or ATP analogues, even at high concentrations of Na+. These results emphasize the existence of multiple functional "E1" and "E2" states in the overall ATPase-ion translocation pathway.

Mechanism of transfer of reduced nicotinamide adenine dinucleotide among dehydrogenases.

The pathway for the transfer of NADH from one dehydrogenase (E1) to another dehydrogenase (E2) has been investigated by studying the E2-catalyzed reduction of S2 by NADH. The experimental conditions are that the concentration of E1 exceeds that of NADH, which in turn is very much greater than E2; hence, the concentration of free (aqueous) NADH is exceedingly low. The rate of reduction of S2 will hence be very slow if unliganded aqueous NADH is required for the E2-catalyzed reaction. Our results with eight dehydrogenases are entirely consistent with the direct transfer of NADH between E1 and E2 whenever the two enzymes transfer hydrogen via opposite faces (A and B) of the nicotinamide ring. Whenever the two enzymes are both A or both B, NADH transfer occurs only via the aqueous solvent. Some mechanistic inferences and their possible physiological significance are discussed.

Molecular basis for the transfer of nicotinamide adenine dinucleotide among dehydrogenases.

NADH is transferred directly from one dehydrogenase enzyme site to another without intervention of the aqueous solvent whenever the two dehydrogenases are of opposite chiral specificity as regards the C4 H of NADH which is transferred in the catalyzed reduction reaction. When both enzymes catalyze the transfer of hydrogen from the same face of the nicotinamide ring, direct enzyme-enzyme transfer of NADH is not possible [Srivastava, D. K., & Bernhard, S. A. (1984) Biochemistry 23, 4538-4545; Srivastava, D. K., & Bernhard, S. A. (1985) Biochemistry (preceding paper in this issue)]. Utilizing an advanced computer graphics facility, and the known three-dimensional coordinates for three dehydrogenases, we have investigated the feasibility of various aspects of the direct transfer of dinucleotide from the site of one enzyme to the site of the other. The facile passage of the coenzyme through the first enzyme site requires an open protein conformation, characteristic of the apoenzyme rather than the holoenzyme structure. Since two dehydrogenases of the same chirality bind coenzyme in the same conformation, the direct transfer of coenzyme from one site to the other is impossible due to the restriction in molecular rotation of the coenzyme in the path of transfer from one binding site to the other; therefore, coenzyme can only be transferred from one dehydrogenase site to another site via the intermediate dissociation of coenzyme into the aqueous milieu. In contrast, when an A dehydrogenase and a B dehydrogenase are juxtaposed, it is stereochemically feasible to transfer the nicotinamide ring from its specific binding site in one enzyme to the site in the other.(ABSTRACT TRUNCATED AT 250 WORDS)

Direct transfer of reduced nicotinamide adenine dinucleotide from glyceraldehyde-3-phosphate dehydrogenase to liver alcohol dehydrogenase.

The reduction of benzaldehyde and p-nitrobenzaldehyde by NADH, catalyzed by horse liver alcohol dehydrogenase (LADH), has been found to be faster when NADH is bound to glyceraldehyde-3-phosphate dehydrogenase (GPDH) than with free NADH. The rate of reduction of aldehyde substrate with GPDH-NADH follows a Michaelian concentration dependence on GPDH-NADH. The reaction velocity is independent of GPDH concentration when [GPDH] greater than [NADH]total. The Km for GPDH-NADH is higher than that for free NADH. The reaction velocities in the presence of excess GPDH over NADH cannot be accounted for on the basis of the free NADH concentration arising from dissociation of the GPDH-NADH complex. These observations suggest that transfer of NADH from GPDH to LADH proceeds through the initial formation of a GPDH-NADH-LADH complex. Arguments for a direct enzyme-coenzyme-enzyme transfer mechanism are substantiated and quantitated both by steady-state kinetic studies and by determinations of all of the appropriate enzyme-coenzyme equilibrium dissociation constants. In contrast, over a similar concentration range, the complex lactate dehydrogenase (LDH)-NADH is not a substrate for the LADH-catalyzed reductions. Likewise, the LADH-NADH complex is not a substrate for the LDH-catalyzed reduction of pyruvate.

Transfer of 1,3-diphosphoglycerate between glyceraldehyde-3-phosphate dehydrogenase and 3-phosphoglycerate kinase via an enzyme-substrate-enzyme complex.

On the basis of the alternatives of direct inter-enzyme transfer vs. dissociation followed by random diffusion, two kinetic models for metabolite transfer between consecutive enzymes are developed. These two models are readily distinguishable experimentally for the transfer of 1,3-diphosphoglycerate (1,3-P2G) between glyceraldehyde-3-phosphate dehydrogenase (GPDH) and 3-phosphoglycerate kinase (PGK). Since 1,3-P2G is exceedingly tightly bound to PGK, the kinetics of its transfer to GPDH are predictably different for each of these two models. Our experiments unambiguously demonstrate that 1,3-P2G is directly transferred between these two enzymes via an enzyme-substrate-enzyme complex. This direct transfer is described by a Michaelis-Menten scheme in which PGK . 1,3-P2G is the "substrate" for GPDH. At high concentrations of PGK . 1,3-P2G, the transfer reaction becomes nearly PGK . 1,3-P2G concentration independent. The rate of the transfer reaction is activated 3.5-fold by saturating quantities of ATP and 20-fold by saturating quantities of 3-PG. Evidence is presented that the PGK . 1,3-P2G complex is structurally distinct from either PGK itself or other PGK . ligand complexes.

Halibut muscle 3-phosphoglycerate kinase. Chemical and physical properties of the enzyme and its substrate complexes.

An efficient procedure for the purification of 3-phosphoglycerate kinase (PGK) from Pacific halibut muscle is described. The molecular weight (43500) and specific activity are similar to those of other species of PGK. The isoelectric point (greater than 9.5) is more than 1.4 pH units higher than that reported for mammalian muscle PGK. The reaction of the seven thiol groups with 5,5'-dithiobis(2-nitrobenzoic acid) (Nbs2) is kinetically biphasic; reaction at a single fast-reacting thiol inactivates the enzyme. The binding of all substrates and products to PGK was observed by 31P NMR. 1,3-Diphosphoglycerate (1,3-P2G) is more tightly bound than is any of the other reaction components. Unlike 1,3-P2G in aqueous solution, the complex with PGK is protected from hydrolysis over a period of weeks. The 31P chemical shifts of this complex are insensitive to pH which suggests that solvent water is excluded from the substrate-bound cleft. As with yeast PGK, the equilibrium constant for the phosphoryl transfer reaction is near unity in the enzyme site environment in contrast to a value of approximately 10(3) (in favor of ATP) in aqueous solution. Since the ternary complex equilibrium 31P NMR spectrum can be accounted for entirely on the basis of the various binary complex spectra, there is no compelling evidence for the involvement of a stoichiometrically substantial phosphoenzyme intermediate.

Role of nicotinamide adenine dinucleotide as an effector in formation and reactions of acylglyceraldehyde-3-phosphate dehydrogenase.

The equilibrium spectral and reactivity properties of a chromophoric acylglyceraldehyde-3-phosphate dehydrogenase (FA-GPDH) have been previously reported. Transient studies of these properties are reported herein. As with true-3-phosphoglyceroyl-enzyme these properties depend on the presence of bound coenzyme (NAD+). The reactivity of the acyl-enzyme toward acceptors (phosphate and arsenate) parallels the extent of its NAD+-induced spectral change [Malhotra, O. P., & Bernhard, S. A. (1973) Proc. Natl. Acad. Sci U.S.A. 70, 2077-2081]. The transcient deacylation of FA-GPDH, preincubated with NAD+, is kinetically biphasic. The relative amplitudes of the fast vs. the slow phase depend on NAD+ concentration but are independent of the nature and concentration of the acyl acceptor. At saturating NAD+ and acceptor concentrations, kinetic biphasicity persists. Perturbation of the acyl-apoenzyme spectrum by NAD+ is also kinetically biphasic. Evidence is presented that the NAD+-requiring acylation of the enzyme results in a protein conformation in which the acyl group is both spectrally perturbed and reactive toward acyl transfer. This acyl-enzyme undergoes a relatively slow isomerization to a conformation in which the acyl spectrum is unperturbed and unreactive in acyl transfer. These two acyl-enzyme conformations are also distinguished by their relative affinities for NAD+; hence, NAD+ is an effector of the conformational equilibrium. Kinetic biphasicity, wherever observed, can be accounted for in terms of two processes: (1) reactivity of the "active" acyl conformation and (2) slow isomerization of the inactive to the active conformation. The two acyl-enzyme conformers are present in finite albeit variable amounts dependent on the extent of NAD+ ligation. Evidence is presented suggesting that each of these conformers has a unique function.

Structural and functional consequences of subunit interactions in glyceraldehyde 3-phosphate dehydrogenase.

A variety of species of GPDH undergo acylation at two of the four active cystein sites per mole of tetrameric enzyme. This reaction requires tightly bound NAD+, a situation restricted to two of the four NAD sites per tetramer. S leads to N acyl transfer from cysteins to lysine in the diacyl enzyme yields an inactive enzyme. The thiol ester bond of acyl enzyme is activated by NAD+ and NADH for the group transfer and reduction reactions, respectively. In furyl acryloyl-GPDH this activation is accompanied by large acyl-spectral shifts, a "blue shift" with NADH and a "red shift" with NAD+. The group transfer reaction as well as spectral shifts show biphasic kinetics. The amplitude of the fast phase of NAD+-induced spectral change in apo-enzyme is equal to that of the fast phase in phosphorolysis (or arsenolysis) at low [NAD+]. The kinetic pattern of spectral shifts by NAD+ and NADH are complementary; the amplitude of the fast phase in one is equal to that of the slow phase in the other. It has been proposed that the acyl enzyme exists in two conformational states. The relative proportion of these states varies with the extent of covalent (acyl group) or non-covalent (NAD+ or NADH) ligation in a manner consistent with the allosteric model of Monod, Wyman and Changeux. These conclusions apply equally to the true substrate acyl enzyme. With 1,3-diphosphoglycerate, a tetra-acylated enzyme is obtained. Two of these four acyl groups react very much faster than the remaining two. A comparison of their specific rates with the steady state turnover numbers indicates that only the less reactive two acyl groups govern the turnover number of the enzyme.