Kinetic Studies of Glyceraldehyde‐3‐Phosphate Dehydrogenase from Rabbit Muscle

Abstract

Initial rate studies at pH 7.6 with three aldehydes, product inhibition patterns with NADH and dead-end inhibition with adenosine diphosphoribose show that the kinetic mechanism of glyceraldehyde-3-phosphate dehydrogenase from rabbit muscle cannot be ordered, and support an enzyme-substitution mechanism. Deviations from Michaelis-Menten behaviour are consistent with negative interactions in the binding of NAD' and instability of the species E(NAD)3 and E(NAD)4. Inhibition with large concentrations of phosphate and arsenate indicates competition for a binding site for glyceraldehyde 3-phosphate, and is not found with glyceraldehyde as substrate.I t is known that the binding of NAD' or NADH to rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (~-glyceraldehyde-3-phosphate : NAD + oxidoreductase, phosphorylating) involves more than one dissociation constant, indicating either that the four subunits of the enzyme are not identical and equivalent, or that the coenzyme binding is negatively cooperative [I -31. The greatest difference of binding free energy is between the second and third coenzyme molecules [3] and there is evidence of a conformational change of the protein at half-saturation with NAD' [4].The steady-state kinetics of the enzyme-catalysed reaction have been investigated in some detail by several groups of workers, with conflicting results and conclusions. Furfine and Velick [5] concluded that mechanism was of the rapid equilibrium, random type involving a quaternary complex, from data with glyceraldehyde 3-phosphate as substrate, whereas Fahien [6], using glyceraldehyde, proposed an ordered mechanism. Orsi and Cleland [7] also proposed an ordered mechanism with NAD', aldehyde and phosphate or arsenate adding in that order, based on studies of inhibition by substrate, products and inactive substrate analogues, and suggested that release of NADH is the final step, rate-limiting when glyceraldehyde 3-phosphate is the substrate. In none of ~ -Ahh,rviarion.s. Gra-3-P, glyceraldehyde 3-phosphate; Gra, Etiqwe. Glyceraldehyde-3-phosphate dehydrogenase (EC glyceraldeh yde. 1.2.1.12).these studies, nor in similar studies with the pigmuscle enzyme [8,9], were deviations from MichaelisMenten behaviour suggestive of negative cooperativity reported. Intersecting patterns of linear reciprocal plots were obtained, which also appeared to eliminate the possibility of an enzyme-substitution mechanism with product dissociation before combination of all the substrates.However, Duggleby and Dennis [lo] have since reported initial rate data for the enzyme from pea seeds consistent with an enzyme-substitution mechanism in which the two stable forms of enzyme involved in the catalytic cycle are an enzyme . NAD' complex and acyl-enzyme. They argued that this is the basic mechanism for the enzyme from other sources also. A similar mechanism had been proposed by Trentham [ll] from pre-steady-state kinetic studies of the enzymes from lobster and sturgeon muscle.The objectives of the present work were to reinvestigate the st...

A major surface protein on group A streptococci is a glyceraldehyde-3-phosphate-dehydrogenase with multiple binding activity.

“…The Km for G-3-P (1.33 mM) in the present investigation is about the same magnitude as was found for GAPDH from B. stearothermophilus (1.0 mM) (46), but 22 times higher than rabbit muscle GAPDH (60/xM) (45) and up to 238 times higher than that reported for GAPDH from human erythrocytes (5.6/tM) (11). Similarly, the Km for NAD (156.7/~M) of SDH was ,,02.4 times that for R stearothermophihs (66 ~M) (46) or only 1.5-4-fold higher than reported for GAPDH enzymes from rabbit muscle (45) and human erythrocytes (11). This finding possibly reflects a conservation within the NAD binding domain, which is located at the NH2 terminus of the GAPDH molecule, and limited conservation within the catalytic domain as reported recently by Ndson et al (49).…”

“…The enzymatic activity of the puri~ed protein was verified by standard assays for GAPDH activity. A specific activity of 34 U/mg of purified protein was found to be comparable to other reported GAPDH enzymes (11,45,46). SDS gel electropho-resis and Western blot analyses of the unhoiled SDH protein sample containing no SDS and saturated with NAD to stabilize the tetrameric structure (47) indicated the presence of a protein band in a position consistent with a molecule of 150 kD, as found for the SDH by molecular sieve chromatography ( Fig.…”

Crystal Structure of Two Ternary Complexes of Phosphorylating Glyceraldehyde-3-phosphate Dehydrogenase from Bacillus stearothermophilus with NAD and d-Glyceraldehyde 3-Phosphate

“…His 176 is postulated to stabilize the tetrahedral intermediate (e) and to facilitate, as an acid (d) or base (e) catalyst, the 1,3-dPG formation. The exchange cofactor step, which consists of NADH release prior to NAD and inorganic phosphate binding, remains controversial (32)(33)(34)(35)(36)(37)(38)(39)(40). RЈ represents the adenine-ribose-phosphate-phosphate-ribose part of the cofactor, NAD.…”

Characterization of the two anion-recognition sites of glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus by site-directed mutagenesis and chemical modification

“…It was thus concluded that the C3-phosphate enhances both the reactivity of aldehyde by increasing the stability of the transition state (Byers, 1977) and the selectivity for the D-stereoisomer of G3P (Byers, 1978;Corbier et al, 1989). However, despite the great number of catalytic (Segal & Boyer, 1953;Duggleby & Dennis, 1974;Meunier & Dalziel, 1978;Canellas & Cleland, 1991;Liu & Huskey, 1992) and energetic studies (Byers, 1977; together with the knowledge of B. stearothermophilus holostructure at 1.8-A resolution, there is no clear evidence as to the individual contribution of Pi and P, sites during the catalytic cycle.…”