Progress in establishing the rate-limiting features and kinetic mechanism of the glyceraldehyde-3-phosphate dehydrogenase reaction

The substrate specificity of sheep liver sorbitol dehydrogenase has been studied by steady-state kinetics over the range pH 7-10. Sorbitol dehydrogenase stereo-selectively catalyses the reversible NAD-linked oxidation of various polyols and other secondary alcohols into their corresponding ketones. The kinetic constants are given for various novel polyol substrates, including L-glucitol, L-mannitol, L-altritol, D-altritol, D-iditol and eight heptitols, as well as for many aliphatic and aromatic alcohols. The maximum velocities (kcat) and the substrate specificity-constants (kcat/Km) are positively correlated with increasing pH. The enzyme-catalysed reactions occur by a compulsory ordered kinetic mechanism with the coenzyme as the first, or leading, substrate. With many substrates, the rate-limiting step for the overall reaction is the enzyme-NADH product dissociation. However, with several substrates there is a transition to a mechanism with partial rate-limitation at the ternary complex level, especially at low pH. The kinetic data enable the elucidation of new empirical rules for the substrate specificity of sorbitol dehydrogenase. The specificity-constants for polyol oxidation vary as a function of substrate configuration with D-xylo > d-ribo > L-xylo > d-lyxo ≈ l-arabino > D-arabino > l-lyxo. Catalytic activity with a polyol or an aromatic substrate and various 1-deoxy derivatives thereof varies with -CH2OH >-CH2NH2 >-CH2OCH3 ≈-CH3. The presence of a hydroxyl group at each of the remaining chiral centres of a polyol, apart from the reactive C2, is also nonessential for productive ternary complex formation and catalysis. A predominantly nonpolar enzymic epitope appears to constitute an important structural determinant for the substrate specificity of sorbitol dehydrogenase. The existence of two distinct substrate binding regions in the enzyme active site, along with that of the catalytic zinc, is suggested to account for the lack of stereospecificity at C2 in some polyols.

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Human glutamic-γ-semialdehyde dehydrogenase. Kinetic mechanism

Biochemical Journal ◽

10.1042/bj2610935 ◽

1989 ◽

Vol 261 (3) ◽

pp. 935-943 ◽

Cited By ~ 11

Author(s):

C Forte-McRobbie ◽

R Pietruszko

Keyword(s):

Kinetic Mechanism ◽

Product Inhibition ◽

Maximal Velocity ◽

Semialdehyde Dehydrogenase ◽

Rate Limiting Step ◽

Dead End ◽

Inhibition Studies ◽

Rate Limiting ◽

Competitive Pattern ◽

Pattern Versus

The kinetic mechanism of homogeneous human glutamic-gamma-semialdehyde dehydrogenase (EC 1.5.1.12) with glutamic gamma-semialdehyde as substrate was determined by initial-velocity, product-inhibition and dead-end-inhibition studies to be compulsory ordered with rapid interconversion of the ternary complexes (Theorell-Chance). Product-inhibition studies with NADH gave a competitive pattern versus varied NAD+ concentrations and a non-competitive pattern versus varied glutamic gamma-semialdehyde concentrations, whereas those with glutamate gave a competitive pattern versus varied glutamic gamma-semialdehyde concentrations and a non-competitive pattern versus varied NAD+ concentrations. The order of substrate binding and release was determined by dead-end-inhibition studies with ADP-ribose and L-proline as the inhibitors and shown to be: NAD+ binds to the enzyme first, followed by glutamic gamma-semialdehyde, with glutamic acid being released before NADH. The Kia and Kib values were 15 +/- 7 microM and 12.5 microM respectively, and the Ka and Kb values were 374 +/- 40 microM and 316 +/- 36 microM respectively; the maximal velocity V was 70 +/- 5 mumol of NADH/min per mg of enzyme. Both NADH and glutamate were product inhibitors, with Ki values of 63 microM and 15,200 microM respectively. NADH release from the enzyme may be the rate-limiting step for the overall reaction.

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Chorismate mutase-prephenate dehydrogenase from Escherichia coli kinetic mechanism of the prephenate dehydrogenase reaction

Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology ◽

10.1016/0167-4838(82)90505-2 ◽

1982 ◽

Vol 702 (2) ◽

pp. 212-219 ◽

Cited By ~ 12

Author(s):

Padmini Sampathkumar ◽

John F. Morrison

Keyword(s):

Escherichia Coli ◽

Kinetic Mechanism ◽

Chorismate Mutase ◽

Prephenate Dehydrogenase ◽

Dehydrogenase Reaction

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Kinetic mechanism of phosphorylase a, II. Isotope exchange studies at equilibrium

Canadian Journal of Biochemistry ◽

10.1139/o70-118 ◽

1970 ◽

Vol 48 (7) ◽

pp. 755-758 ◽

Cited By ~ 18

Author(s):

H. D. Engers ◽

W. A. Bridger ◽

N. B. Madsen

Keyword(s):

Exchange Rates ◽

Isotope Exchange ◽

Initial Rate ◽

Kinetic Mechanism ◽

Rabbit Muscle ◽

Glucose Inhibition ◽

Rate Limiting Step ◽

Equilibrium Exchange ◽

Muscle Phosphorylase ◽

Rate Limiting

In order to confirm the kinetic mechanism which was proposed for rabbit muscle phosphorylase a on the basis of initial rate studies and UDP-glucose inhibition experiments, isotope exchange studies at equilibrium were performed, both in the presence and absence of the modifier AMP.Both the 14C-glucose [Formula: see text] and the [Formula: see text]1-phosphate equilibrium exchange rates increased to a maximum as the concentrations of the varied substrates became saturating, either in the presence or absence of AMP. The plateaus observed in these experiments indicate the lack of inhibition of the exchange of one pair of substrates when the concentration of the other substrate pair was raised, and confirms the proposed random addition of substrates to the enzyme.The fact that similar exchange rates were observed for either reaction direction reinforced the concept that rapid equilibrium conditions apply to the phosphorylase a mechanism; i.e. the interconversion of the ternary complexes tends to be the rate-limiting step in the reaction sequence.Maximal velocities determined from initial rate data reported in the previous paper agreed with those calculated from isotope exchange rates.

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Kinetic studies of formate dehydrogenase

Biochemical Journal ◽

10.1042/bj1200763 ◽

1970 ◽

Vol 120 (4) ◽

pp. 763-769 ◽

Cited By ~ 27

Author(s):

D. Peacock ◽

D. Boulter

Keyword(s):

Carbon Dioxide ◽

Formate Dehydrogenase ◽

Kinetic Studies ◽

Kinetic Mechanism ◽

Product Inhibition ◽

Reverse Reaction ◽

Enzyme Form ◽

Rate Limiting Step ◽

Measurable Rate ◽

Rate Limiting

1. The kinetic mechanism of formate dehydrogenase is a sequential pathway. 2. The binding of the substrates proceeds in an obligatory order, NAD+ binding first, followed by formate. 3. It seems most likely that the interconversion of the central ternary complex is extremely rapid, and that the rate-limiting step is the formation or possible isomerization of the enzyme–coenzyme complexes. 4. The secondary plots of the inhibitions with HCO3− and NO3− are non-linear, which suggests that more than one molecule of each species is able to bind to the same enzyme form. 5. The rate of the reverse reaction with carbon dioxide at pH6.0 is 20 times that with bicarbonate at pH8.0, although no product inhibition could be detected with carbon dioxide. The low rate of the reverse reaction precluded any steady-state analysis as the enzyme concentrations needed to obtain a measurable rate are of the same order as the Km values for NAD+ and NADH.

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