Srinivasan R
Department of Chemistry/Physics, Uniersity of Alaska, Anchorage 99508.
Biochim Biophys Acta. 1991 Jan 23;1073(1):18-22. doi: 10.1016/0304-4165(91)90177-i.
The reductive amination of alpha-ketoglutarate, catalyzed by bovine liver glutamate dehydrogenase, is inhibited by various anions. Formate and acetate ions are competitive with alpha-ketoglutarate. The pH dependence of the pKi profiles for these anions reveals that they bind to the enzyme-NADPH complex only when an enzymatic residue of pK 8.0 +/- 0.1 in the binary complex is protonated. The ionization of this residue has a delta Hion of 15 +/- 4 kcal/mol. These pK and delta Hion values are not significantly different from those observed in the same complex for the enzyme group which binds the gamma-CO2- of alpha-ketoglutarate and oxalylglycine. It is concluded that formate and acetate also bind to the gamma-carboxylate site in enzyme-NADPH. The Ki values for formate and acetate in a buffer containing 0.1 M phosphate are 20 +/- 4 and 32 +/- 5 mM, respectively, when the pK 8.0 group is fully protonated. Phosphate and trifluoroacetate also show an inhibitory effect, while valerate and sulfate have little effect on the reductive amination rates. The results suggest that specific anions can bind to the gamma-carboxylate site by ionic interactions and alter the kinetic and thermodynamic parameters of the glutamate dehydrogenase-NADPH complex in significant ways.
牛肝谷氨酸脱氢酶催化的α-酮戊二酸还原胺化反应受到多种阴离子的抑制。甲酸根离子和乙酸根离子与α-酮戊二酸存在竞争性。这些阴离子的pKi曲线对pH的依赖性表明，只有当二元复合物中pK为8.0±0.1的酶残基质子化时，它们才会与酶-NADPH复合物结合。该残基的电离具有15±4千卡/摩尔的ΔHion。这些pK和ΔHion值与在结合α-酮戊二酸和草酰甘氨酸γ-CO2-的酶基团的同一复合物中观察到的值没有显著差异。可以得出结论，甲酸根离子和乙酸根离子也结合到酶-NADPH中的γ-羧酸盐位点。当pK 8.0基团完全质子化时，在含有0.1 M磷酸盐的缓冲液中，甲酸根离子和乙酸根离子的Ki值分别为20±4和32±5 mM。磷酸根离子和三氟乙酸根离子也表现出抑制作用，而戊酸根离子和硫酸根离子对还原胺化速率影响很小。结果表明，特定的阴离子可以通过离子相互作用结合到γ-羧酸盐位点，并以显著方式改变谷氨酸脱氢酶-NADPH复合物的动力学和热力学参数。
