Determination of the Kinetic and Chemical Mechanism of Malic Enzyme Using (2R,3R)-erythro-Fluoromalate as a Slow Alternate Substrate

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• 作者：Jeffrey L. Urbauer ; Debra E. Bradshaw ; and W. W. Cleland
• 刊名：Biochemistry
• 出版年：1998
• 出版时间：December 22, 1998
• 年：1998
• 卷：37
• 期：51
• 页码：18026 - 18031
• 全文大小：50K
• 年卷期：v.37,no.51(December 22, 1998)
• ISSN：1520-4995

文摘

(2R,3R)-erythro-Fluoromalate, but not the threo isomer, is a slow substrate for chicken livermalic enzyme with either NADP or 3-acetylpyridine-NADP (APADP) as the other substrate. The K_m forerythro-fluoromalate is similar to that of malate, but the turnover number with NADP is 3300-fold slower,although 5.5-fold faster with APADP than with NADP. Deuteration of fluoromalate at C-2 gave an isotopeeffect on V/K of 1.39 with NADP and 3.32 with APADP. With NADP, the ¹³C isotope effects at C-4were 1.0490 with unlabeled and 1.0364 with deuterated fluoromalate. With APADP, the correspondingvalues were 1.0138 and 1.0087. These data show that the mechanism is stepwise with both nucleotidesubstrates, in contrast to the reaction of malate and APADP, which was postulated to be concerted byKarsten et al. [Karsten, W. E., and Cook, P. F. (1994) Biochemistry 33, 2096-2103], a conclusion recentlyshown to be correct by Edens et al. [Edens, W. A., Urbauer, J. L., and Cleland, W. W. (1997) Biochemistry36, 1141-1147]. To explain the effect of deuteration on the ¹³C isotope effect with APADP, it is necessaryto assume a secondary ¹³C isotope effect at C-4 on the hydride transfer step of ~1.0064 (assuming 5.7 asthe intrinsic primary deuterium isotope effect and 1.054 as the product of the ¹³C equilibrium isotopeeffect on hydride transfer and the intrinsic ¹³C isotope effect on decarboxylation). The secondary ¹³Cisotope effect on hydride transfer is thought to result from hyperconjugation between the carbonyl groupand C-4 of the enzyme-bound fluorooxaloacetate intermediate.