The Mechanism of Oxidation of Aldehydes by Glyceraldehyde-3-Phosphate Dehydrogenase

Racker, Efraim; Krimsky, I.

doi:10.1016/s0021-9258(18)55530-7

Cited by 300 publications

(23 citation statements)

References 20 publications

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“…Apoenzyme was prepared by activated charcoal treatment (Racker & Krimsky, 1952). Protein concentrations were determined spectrophotometrically, using molar extinction coefficients of 1.17 X 105 M-1 cm-1 and 1.31 X 105 M-1 cm-1 for B. stearothermophilus apo-and holoenzymes, respectively.…”

Section: Methodsmentioning

confidence: 99%

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

Corbier

Michels²,

Wonacott³

et al. 1994

Biochemistry

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The active site of the glycolytic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) contains two anion recognition sites which have been attributed to the phosphate binding of the substrates, namely, glyceraldehyde 3-phosphate (Ps site) and inorganic phosphate (Pi site) [Moras et al. (1975) J. Biol. Chem. 250, 9137-9162]. In order to probe the role of both sites during the catalytic event, Arg 195 from the Pi site and Arg 231 from the Ps site of the Bacillus stearothermophilus enzyme have been changed to Leu and Gly, respectively, by site-directed mutagenesis. A comparative study of the chemical reactivity of the mutants and wild type toward 2,3-butanedione revealed a similarly high reactivity only for the R195L mutant and wild type, suggesting that only Arg 231 is chemically reactive toward 2,3-butanedione and that its reactivity is not influenced by the presence of the residue Arg 195, which is only 4 A distant. The kinetic consequences of the mutations were also analyzed for the consecutive steps in the forward catalytic reaction. The replacement of Arg 195 by Leu leads to a marked decrease of the rate of the first steps of the reaction which lead to the acylenzyme formation, in particular, the rate of enzyme-substrate association, while these steps occur at a similar or higher rate when Arg 231 is replaced by Gly. Furthermore, the mutations R195L and R231G also result in a 550-fold and 16,400-fold decrease in the second-order rate constant of phosphorolysis. This step becomes rate-determining for the R195L mutant.(ABSTRACT TRUNCATED AT 250 WORDS)

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Section: Methodsmentioning

confidence: 99%

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

Corbier

Michels²,

Wonacott³

et al. 1994

Biochemistry

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“…Spectrophotometric Properties of the Enzyme Analog Complexes. Racker and Krimsky (1952) first showed that the binding of /3-NAD to the enzyme produces a broad absorbance band with a maximum at 360 µ. Figure 1 shows the increase in absorbance at 360 µ produced by successive additions of the two analogs which gave this effect.…”

Section: Resultsmentioning

confidence: 98%

“…Previous reports have concluded that the principal form of /3-NAD in aqueous solution at neutral pH has the aromatic rings stacked (Sarma and Kaplan, 1968Kaplan, , 1970aSarma et al. 1970) and that the reduced coenzyme when bound to GPDH is in a conformation with the aromatic rings stacked (Velick, 1958(Velick, , 1961.…”

Section: Discussionmentioning

confidence: 99%

“…According to Sarma andKaplan (1968, 1970a) acetylpyridine-AD exists in a stacked conformation, but, unlike /3-NAD, the acetylpyridine ring is in the anti rather than the syn conformation with resulting changes in the conformation of the PPR backbone. Deamino-NAD occurs primarily in the open conformation (Sarma and Kaplan, 1968Kaplan, , 1970a. The unusually high Km for this analog and its failure to stabilize the enzyme suggest that only the stacked conformation is active as a substrate.…”

Section: Discussionmentioning

confidence: 99%

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Interaction of nicotinamide adenine dinucleotide and its analogs with glyceraldehyde 3-phosphate dehydrogenase

D¹,

Me²

1971

Biochemistry

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“…Early studies of the rabbit muscle enzyme (Racker & Krimsky 1952;Segal & Boyer 1953) indicated an enzyme-substitution mechanism of catalysis (figure 1) involving the intermediate formation of 3-phosphoglyceryl-enzyme acylated at its iodoacetate-sensitive thiol groups. The other stable enzyme species in this double-displacement mechanism was assumed to be the binary complex of enzyme with NAD+ (holoenzyme) because the recrystallized enzyme retains a complement of firmly bound NAD+.…”

Section: Pathway Of Catalysismentioning

confidence: 99%

Glyceraldehyde-3-phosphate dehydrogenase

Dalziel

McFerran

Wonacott

1981

Phil. Trans. R. Soc. Lond. B

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Conflicting experimental evidence of the pathway of catalysis for the enzyme from rabbit, pig and lobster muscle tissues is reviewed. Transient kinetic studies with the enzyme from rabbit muscle are presented. The results are shown to be consistent with the double-displacement mechanism of catalysis originally proposed by Segal & Boyer (1953). The rate constant for combination of the aldehyde form of the substrate with the NAD+ complex of the enzyme is about 3 X 10(7) M-1 S-1, and for all four subunits of the molecule the rate constant for hydride transfer in the ternary complex formed is greater than 10(3) S-1, consistent with their simultaneous participation in catalysis. Recent steady-state kinetic studies with the rabbit muscle enzyme, in contrast to earlier studies, also provide evidence to support the Segal-Boyer pathway if the kinetic effects of the negative cooperativity of NAD+ binding are taken into account. Experimental data for the binding of NAD+ to the enzyme from muscles and from Bacillus stearothermophilus, and their interpretations, are also briefly reviewed. The information currently available from X-ray crystallography regarding the structures of holoenzyme and apoenzyme from B. stearothermophilus and lobster muscle is outlined.

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The Mechanism of Oxidation of Aldehydes by Glyceraldehyde-3-Phosphate Dehydrogenase

Cited by 300 publications

References 20 publications

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

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

Interaction of nicotinamide adenine dinucleotide and its analogs with glyceraldehyde 3-phosphate dehydrogenase

Glyceraldehyde-3-phosphate dehydrogenase

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