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The kinetics and stoichiometry of the reaction of sturgeon muscle glyceraldchyde-3-PO 4 -dehydrogenase (GPDH) with the disulfide interchange reagent bis(2,2' dithio-bis(5-nitrobenzoate) (DTNB) has been studied in detail. The native enzyme, a tetramer of covalently identical subunits, reacts relatively rapidly with precisely four equivalents of reagent, although there are three cysteine residues per subunit (12 per tetratner). Reaction of these four cystcines leads to total catalytic inactivation; the extent of inactivation is proportional to the fractional reaction. The rate of reaction is dependent on the extent of bound NAD: reactivity being very much greater at unliganded sites. The reaction with apo-enzyme is fastest, bimolecular and monophasic. Over a wide range of NAD concentration, however, the reaction of enzyme with a large molar excess of reagent is precisely biphasic, and each individual kinetic experiment can be analytically described by two pseudo first-order (NAD concentration-dependent) rate constants and two unequal NAD concentration-insensitive amplitudes. The biphasicity in rate is quantitatively explainable on the basis of a C 2 symmetry for the tetrameric subunits with a tighter binding of NAD at two of the four sites, if high reactivity is exclusively dependent on the absence of bound NAD. The inequality in the two amplitudes, however, requires either a more complex or a more dynamic model. Arguments are presented for the appropriateness of a C 2 symmetry model in which intramolecular transconformational isomerization of tight and loose NAD binding sites is possible. The equilibrium constant for the isomerization is estimable from the macroscopic specific rates and amplitudes. This “flip-over” C 2 symmetry model is apropos to all situations of negative cooperativity in ligand binding to tetramers, as is discussed.

A 3 A resolution Fourier map of the glycolytic enzyme 3-phosphoglycerate kinase from horse muscle, has been calculated using a single isomorphous derivative. The map is interpretable and has been used to define the organization of the molecule's single polypeptide chain. The enzyme has about 355 amino acid residues, indicating that the molecular weight is close to 38,000. The molecule is highly structured with more than half its residues organized into 11α-helical segments and two large β-sheet structures. The binuclear structure of the enzyme has been confirmed by the highresolution map. The two lobes or domains are clearly structurally independent and physically separated except in the neck region, which is now found to be composed of two chains running in opposite directions. Each domain is organized around a central β-sheet core, which in domain A (the ADP ATP binding domain) is composed of six parallel strands, while that in domain B has five strands in which three parallel strands are followed by two running in the opposite direction. On either side of these two sheets lie most of the α-helical segments, which have a tendency to be aligned parallel with the strands of the sheets. The enzyme co-factors, Mg-ADP and Mg-ATP, have been located in low-resolution difference maps. They are both located at a binding site on domain A, which consists of a slot for the adenine ring at the extremity of the parallel β-sheet. The metal in the ADP complex has been defined, and it appears to be linked to both the α and β-phosphates and an enzyme side-chain. The phosphate chain of the co-factors is located in the space between domain A and domain B, with the γ-phosphate of ATP near a side-chain from domain B. The position of the γ-phosphate, which is transferred during the enzymatic reaction, can be interpreted as supporting a mechanism involving a phosphoenzyme intermediate and suggesting that the active site of the enzyme is divided between the two domains, with domain A carrying the ADP ATP binding site and domain B the phosphoglycerate binding site and the catalytic site. Most remarkably, the structure of domain A bears a strong resemblance to the common nucleotide binding unit that has been found in four dehydrogenases. The location of the ADP ATP binding site on this unit is in an equivalent position to the NAD binding site in the dehydrogenases. This relationship is discussed.

Eight n-alkylammonium chlorides were demonstrated to inhibit the l -α-glycerophosphate dehydrogenase-catalyzed oxidation of l -α-glycerophosphate. The inhibition obtained was shown to be competitive with respect to the coenzyme, NAD. Interactions of these inhibitors with a “pyridinium ring” region at the NAD binding site of the enzyme was suggested by the competitive nature of the inhibition obtained and by the simultaneous binding of n-decylammonium chloride and adenylic acid to the enzyme. The binding of n-alkylammonium chlorides to l -α-glycerophosphate dehydrogenase increases with increasing chain length of the inhibitor, indicating non-polar interactions with a hydrophobic region of the enzyme to be of importance in these binding processes.

Multiple inhibition studies of yeast alcohol dehydrogenase (alcohol:NAD oxidoreductase, E.C. I.I.I.I) were carried out using as inhibitor pairs various combinations of n -alkylammonium chlorides with adenylic acid and adenosine diphosphoribose, and N 1 -alkylnicotinamide chlorides with adenylic acid and adenosine diphosphoribose. Inhibitors interacting with the enzyme at the “pyridinium ring” region of the NAD binding site were demonstrated to be bound to the enzyme simultaneously with adenine derivatives. A pronounced positive interaction was observed in the simultaneous binding of adenine and nicotinamide derivatives, suggesting a possible ring-ring interaction in the positioning of these inhibitors on the enzyme. N 1 -Alkyl-nicotinamide chlorides are bound simultaneously with NADH to yeast alcohol dehydrogenase and do not effectively inhibit the enzyme-catalyzed reduction of acetaldehyde. The studies presented suggest a different mode of binding to yeast alcohol dehydrogenase for the oxidized and reduced pyridine moieties of the coenzymes, NAD and NADH.

A method is described for the isolation of glyceraldehyde 3-phosphate dehydrogenase from rabbit liver. The enzyme has been crystallized as the NAD complex and its chemical and physical properties have been compared with those of the muscle enzyme. The kinetics of the reversible reaction catalyzed by the dehydrogenases is sensitive to temperature and ionic strength and has been examined at 37°, pH 7.4, in a solvent of ionic strength 0.1. Under these conditions the outstanding new feature of the reverse reaction, the reductive dephosphorylation of 3-phosphoglyceroyl phosphate, is the positive cooperativity of the response of both enzymes to concentration increments of the acyl phosphate and the increase in this cooperativity as a function of concentration of the cosubstrate, NADH. At low concentrations of the acyl phosphate, the reduced coenzyme exerts an inhibitory function which is abolished by high concentrations of acyl phosphate or by low concentrations of NAD acting as a heterotropic effector. The same concentrations of NAD which activate at low acyl phosphate concentration inhibit at high concentrations of the 3-carbon substrate by competing with NADH. Activation of the reaction by NAD involves a transition of the acyl phosphate saturation function from a sigmoidal to the hyperbolic form and hence sensitizes the enzyme to lower concentrations of acyl phosphate. The substrate inhibition by NADH and the activation by NAD may be accounted for by the binding of the nucleotides to catalytic sites of the tetrameric protein that are not occupied by the acyl enzyme intermediate. Although the competitive advantage in binding to the apoprotein strongly favors NAD over NADH, the competition under reaction conditions tends to be equalized at nonacylated sites of the acylated protein and is drastically reversed at the acylated sites. Thus at kinetic saturation levels of the acyl phosphate Knadh remains in the 10-µm range, but half-maximal inhibition by NAD occurs at concentrations of the latter that are in excess of 1000 µm. These properties involve both ligandinduced conformational transitions of the protein and a local steric effect of the acyl group that interferes with the binding of the oxidized but not the reduced form of the pyridine nucleotide. In the forward or oxidative phosphorylation reaction the reciprocal kinetic plots are entirely linear except under conditions of product inhibition by NADH. The preferential binding of NADH at an acylated site is not strongly expressed in the inhibition by added NADH because such sites are already occupied by product NADH that is released subsequent to the rate-limiting acyl group transfer from enzyme to orthophosphate ion. The acyl phosphate is a strong inhibitor of the forward reaction, noncompetitive with NAD and strictly competitive with aldehyde. Intracellularly, the enzyme always operates in the presence of high concentrations of NAD which are required to provide the driving force for the energy-conserving reaction. Functional reversibility, under the adverse substrate concentration distributions in the aerobic hepatocyte, is maintained by regulatory effects of NAD binding which promote the reaction of extremely low concentrations of acyl phosphate, and by the differential effects of acylation of the protein which allow access of substrate NADH and at the same time eliminate a prohibitive inhibition by the high concentrations of NAD. The catalytic properties of the liver and muscle enzyme are qualitatively similar but exhibit some quantitative differences that would favor the more diversified metabolic requirements of liver. However, no definitive structural differences have been identified.

The absorption spectrum of an activesite specific chromophoric acyl enzyme, sturgeon β-(2-furyl)-acryloyl-glyceraldehyde-3-phosphate dehydrogenase, is reported. This acyl enzyme undergoes all of the catalyzed reactions characteristic of the intermediate of the physiological acyl enzyme, 3-phospho-D-glyceroyl-glyceraldehyde-3-phosphate dehydrogenease. The rates of reactions of both these acyl enzymes depend strongly on the extent of interaction of the acyl enzyme with the oxidized coenzyme, NAD + , even where the “redox” properties of the coenzyme are not required. Likewise, the spectral properties of chromophoric acyl enzyme are affected by the extent of bound NAD. Under the pseudophysiological conditions reported herein, there is a stoichiometric limitation of two furylacryloyl-acyl groups per enzyme molecule containing four covalently-equivalent subunits. The binding of NAD both to the apoenzyme and to the diacyl enzyme is heterogeneous: at low extents of NAD occupancy, NAD binding is stronger. The binding to acyl enzyme can be quantitatively described by an enzyme model involving a tetramer with 2-fold symmetry, and consequently containing equal numbers of two classes of sites. NAD binding to difurylacryloyl-enzyme occurs virtually discretely, first to the two unmodified (tight-binding) sites, followed by looser binding to the two acyl-sites. NAD occupancy at these latter sites transforms the chromophoric acyl spectrum from that characteristic of a model furylacryloyl-thiol ester in H 2 O to a highly perturbed furylacryloyl spectrum characteristic of monomeric native “active-thiol” furylacryloyl-enzymes. Likewise the acyl reactivity towards arsenolysis depends on the extent of NAD bound to the loose sites. Elimination of the tight binding of NAD to the difurylacryloyl enzyme tetramer by alkylation of the remaining two free SH groups with iodoacetate has no apparent influence on the NAD-dependent furylacryloyl-spectral perturbation at the “two equivalent acyl sites,” even though it eliminates the apparent “negative cooperativity” in NAD binding.

Acetoacetyl-CoA was found to strongly inhibit the dehydrogenation of L-3-hydroxybutyryl-CoA catalyzed by L-3-hydroxyacyl-CoA dehydrogenase from pig heart. The inhibition constant (Ki) was determined to be 7.7 × 10−6 M, a value which is similar to the Km value of 12 × 10−6 M obtained for acetoacetyl-CoA in its NADH-dependent reduction catalyzed by the same enzyme. A suggested ordered BiBi mechanism for this enzyme, with NAD binding to the enzyme first, explains the observed noncompetitive nature of this inhibition. The possible effect of this inhibition on fatty acid oxidation is discussed.

The crystal structure of lobster d -glyceraldehyde-3-phosphate dehydrogenase has been analyzed. A 3.0 A resolution electron density map of an averaged subunit showed that: 1. (1) The subunit structure consists of two domains. The first (residues 1 to 149) is essentially the coenzyme binding fold whereas the second (residues 150 to 334) provides residues for catalysis, specificity and subunit-subunit co-operativity. 2. (2) The first domain is similar to structures found in other dehydrogenases and kinases. Some of the important residues involved in binding the coenzyme, NAD+, are the same in d -glyceraldehyde-3-phosphate dehydrogenase as in lactate dehydrogenase. 3. (3) The conformation of NAD+ is essentially the same in d -glyceraldehyde-3-phosphate dehydrogenase as in lactate dehydrogenase, apart from a 180 ° rotation about the ribose-nicotinamide glycosidic bond, thus exposing the B side of the nicotinamide ring to the substrate. 4. (4) The second domain contains histidine 176 situated near the essential cysteine 149 and presumably acting as a base during catalysis. 5. (5) Lysine 183 binds to the pyrophosphate in the active site of the adjacent subunit, creating the unique situation in which the catalytic center contains residues from two different subunits. Thus the NAD binding sites are directly linked in pairs by virtue of a molecular 2-fold axis, forming a functional dimer. The co-operativity (positive in yeast and negative in muscle enzyme) and the half-of-the-sites reaction properties are probably associated with this characteristic. 6. (6) The position of the two sulfate anion sites suggests the position of binding of the substrate phosphate and the inorganic phosphate to be subsequently incorporated into the product. 7. (7) The amino acids in the active center region and involved in subunit-subunit contacts are more highly conserved than other residues.

At pH 9.0 the activity of crystalline glutamate dehydrogenase was increased by the addition to the incubation mixture of various electrolytes including sodium chloride, potassium phosphate, Tris, 2-amino-2-methyl-1, 3-propanediol and methionine. Potassium phosphate was most stimulatory and, at an optimum concentration, increased activity three to four-fold. No stimulation was obtained at pH 7.6. Potassium phosphate and the other electrolytes abolished activation by high concentrations of NAD or NADP and altered the inhibitory effects obtained by compounds which presumably do not interact at the NAD binding site. These electrolytes also overcame the inhibition by GTP, a ligand which binds at the same site as NAD, but they did not suppress the reactivity with ADP. It is proposed that at pH 9.0 potassium phosphate and other electrolytes alter glutamate dehydrogenase so that the regulatory site which binds NAD or GTP is no longer available to these ligands.

The binding of NAD+ to rabbit-muscle glyceraldehydephosphate dehydrogenase (D-glyceraldehyde 3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12) has been studied extensively by several groups (1, 2, 3, 4). Since, however, a start has been made with determining the tertiary and quaternary structure of the lobster-muscle enzyme (5) by X-ray crystallographic data, and its primary structure has been elucidated (6), it was important to extend our binding studies to this enzyme (7).

Changes induced on addition of the cofactor, NAD, to glyceraldehyde-3-phosphate dehydrogenase from yeast have been investigated by hydrodynamic and spectroscopic techniques. As shown by light-scattering and sedimentation and diffusion studies, the tetrameric structure of the holoenzyme persists under all but strong dissociating conditions: there is thus no change in the quaternary structure on binding. On the other hand, binding is accompanied by an increase of some 4% in the sedimentation coefficient, which is too large to be explicable in terms of the increase in particle weight by bound ligand. A conformational change is also observed by optical rotatory dispersion and circular dichroism measurements. The magnitude of the Cotton effect associated with the aromatic chromophores of the protein, near 280 nm, increases, whereas the rotation in the neighbourhood of 233 nm undergoes a small diminution as the cofactor is added. It is shown that these changes are conformational in origin, rather than a consequence of NAD attachment per se. Increasing the temperature in the range 20–40° leads to a transition from non-cooperative to cooperative binding of NAD to the apoenzyme. At 40°, where NAD binding is weakly sigmoidal, the change in optical rotation is likewise sigmoidal and parallels the increase in the sedimentation coefficient. At 20° both the uptake of NAD and the structural changes are hyperbolic. In the cooperative situation the data are shown to be consistent with the two-state “concerted” model for ligand-binding and incompatible with a sequential mechanism.

The objects of investigations were single crystals of glyceraldehyde 3-phosphate dehydrogenase. Precession X-ray diffraction patterns of several reciprocal lattice planes were obtained. The specifity of the molecular packing in crystals provided a direct possibility to find tetrahedral symmetry of a holo-enzyme molecule, while the decrease of the crystal symmetry during conversion from holo- to apo-form suggests the decrease of glyceraldehyde 3-phosphate dehydrogenase molecular symmetry during this process. This result is of interest in connection with the symmetry transformations of other dehydrogenases during NAD binding.