Reaction of glyceraldehyde 3-phosphate dehydrogenase with aliphatic aldehydes

Fife, Thomas H.; Rikihisa, Tadaaki

doi:10.1021/bi00823a006

Cited by 13 publications

(3 citation statements)

References 15 publications

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“…No adjustment has been made to Km. The references were as follows: (a) Harting & Velick (1954); (b) Nygiiard & Sumner (1952); (c) Fife & Rikihisa (1970); (d) Vdick & Hayes (1953); (e) Ferdinand (1964). Aldehyde R V,.…”

Section: Nature Of the Aldehyde-enzyme Complexmentioning

confidence: 99%

The reactions of D-glyceraldehyde 3-phosphate with thiols and the holoenzyme of D-glyceraldehyde 3-phosphate dehydrogenase and of inorganic phosphate with the acyl-holoenzyme

Armstrong

Trentham

1976

Biochemical Journal

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D-Glyceraldehyde 3-phosphate forms adducts with thiols. These adducts, which are presumed to be hemithioacetals, equilibrate rapidly with the unhydrated form of the aldehyde, which is the subtrate for D-glyceraldehyde 3-phosphate dehydrogenase. The adduct provides a substrate buffer system whereby a constant low free aldehyde concentration can be maintained during the oxidation of aldehyde by the enzyme and NAD+. With this system, the kinetics of the association of the aldehyde with the enzyme were examined. The rate profile for this reaction is a single exponential process, showing that all four active sites of the enzyme have equivalent and independent reactivity towards the aldehyde, with an apparent second-order rate constant of 5 X 10(7)M-1-S-1 at pH8.0 and 21 degrees C. The second-order rate constant becomes 8 X 10(7)M-1-S-1 when account is taken of the forward and reverse catalytic rate constants of the dehydrogenase. The pH-dependence of the observed rate constant is consistent with a requirement for the unprotonated form of a group of pK 6.1, which is the pK observed for second ionization of glyceraldehyde 3-phosphate. The rate of phosphorolysis of the acyl-enzyme intermediate during the steady-state oxidative phosphorylation of the aldehyde was studied, and is proportional to the total Pi concentration up to at least 1 mM-Pi at pH 7.5. The pH-dependence of the rate of NADH generation under these conditions can be explained by the rate law d[NADA]/dt = k[acy] holoenzyme][PO4(3-)-A1, where thioester bond, although kinetically indistinguishable rate equations for the reaction are possible. The rates of the phosphorolysis reaction and of the aldehyde-association reaction decrease with increasing ionic strength, suggesting that the active site of the enzyme has cationic groups which are involved in the reaction of the enzyme with anionic substrates.

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Section: Nature Of the Aldehyde-enzyme Complexmentioning

confidence: 99%

The reactions of D-glyceraldehyde 3-phosphate with thiols and the holoenzyme of D-glyceraldehyde 3-phosphate dehydrogenase and of inorganic phosphate with the acyl-holoenzyme

Armstrong

Trentham

1976

Biochemical Journal

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“…Aldehyde Reactivity. The aldehydes examined in this study are much poorer substrates for glyceraldehyde-3-phosphate dehydrogenase than is D-glyceraldehyde 3-phosphate (Fife & Rikihisa, 1970;Byers, 1978). In order to minimize inactivation of the enzyme by nonspecific reaction of the aldehydes with amino groups on the protein, and to minimize the nonenzymatic reaction of the aldehydes with NAD (which results in formation of an adduct which absorbs at 340 nm) (Byers, 1978), we carried out the reactions at low aldehyde concentrations (<0.01 M).…”

Section: Methodsmentioning

confidence: 71%

“…The dependence of the second-order rate constant (k^JWM°°r) on * is characterized by a p* value of 2.81. Fife & Rikihisa (1970) found a dependence of kcat on * for the reaction of a series of aliphatic aldehydes with rabbit muscle glyceraldehyde-3-phosphate dehydrogenase, characterized by a p* value of 2.08. There is not as good a correlation between KM and * as there is between /ceat and * for these aldehydes.…”

Section: Discussionmentioning

confidence: 98%