Miziorko H M, Lane M D
J Biol Chem. 1977 Feb 25;252(4):1414-20.
Acetyl-CoA reacts stoichiometrically with a cysteinyl sufhydryl group of avian liver 3-hydroxy-3-methylglutaryl (HMG)-CoA synthase to yield acetyl-S-enzyme (Miziorko H.M., Clinkenbeard, K.D., Reed, W.D., and Lane, M.D. (1975) J. Biol. Chem. 250, 5768-5773). Evidence that acetyl-S-enzyme condenses with the second substrate, acetoacetyl CoA, to form enzyme-S-HMG-SCoA has been obtained by trapping and characterizing this putative intermediate. [14C]Acetyl-S-enzyme was incubated briefly at -25 degrees with acetoacetyl-CoA, precipitated with trichloroacetic acid, and the labeled acylated enzyme species were isolated. Performic acid oxidation of the precipitated [14C]acyl-S-enzyme intermediates produced volatile [14C]acetic acid from unreacted [14C]acetyl-S-enzyme and nonvolatile [14C]3-hydroxy-3-methyl glutaric acid from enzyme-S-[14C]HMG-SCoA. Condensation of unlabeled acetyl-S-enzyme with [14C]aceto-acetyl-CoA or acetoacetyl-[3H]CoA also produced labeled enzyme-S-HMG-SCoA. Thus, the acetyl moiety from acetyl-CoA and the acetoacetyl and CoA moieties from acetoacetyl-CoA all are incorporated into the HMG-CoA which is covalently-linked to the enzyme. Enzyme-S-[14C]HMG-SCoA was subjected to proteolytic digestion under conditions favorable for intramolecular S to N acyl transfer in the predicted cysteine-S-[14C]HMG-SCoA fragment. Performic acid oxidation of the protease-digested material yields N-[14C]HMG-cysteic acid indicating that HMG-CoA had been covalently bound to the enzyme via the -SH of an active site cysteine. An isotope trapping technique was employed to test the kinetic competence of acetyl-S-enzyme as an intermediate in the HMG-CoA synthase-catalyzed reaction. Evidence is presented which indicates that the rate of condensation of acetoacetyl-CoA with acetyl-S-enzyme to form enzyme-S-HMG-SCoA is more rapid than either the acetylation of the synthase by acetyl-CoA or the overall forward reaction leading to HMG-CoA. These observations, together with indirect evidence that hydrolysis of enzyme-S-HMG-SCoA is extremely rapid, suggest that acetylation of synthase is the rate-limiting step in HMG-CoA synthesis.
乙酰辅酶A与禽肝3-羟基-3-甲基戊二酰（HMG）-辅酶A合酶的一个半胱氨酰巯基按化学计量反应，生成乙酰-S-酶（米齐奥科H.M.、克林肯比尔德K.D.、里德W.D.和莱恩M.D.（1975年）《生物化学杂志》250卷，5768 - 5773页）。通过捕获和鉴定这种假定的中间体，已获得证据表明乙酰-S-酶与第二种底物乙酰乙酰辅酶A缩合形成酶-S-HMG-SCoA。将[¹⁴C]乙酰-S-酶与乙酰乙酰辅酶A在-25℃短暂孵育，用三氯乙酸沉淀，然后分离标记的酰化酶种类。沉淀的[¹⁴C]酰基-S-酶中间体经过甲酸氧化，未反应的[¹⁴C]乙酰-S-酶产生挥发性的[¹⁴C]乙酸，而酶-S-[¹⁴C]HMG-SCoA产生非挥发性的[¹⁴C]3-羟基-3-甲基戊二酸。未标记的乙酰-S-酶与[¹⁴C]乙酰乙酰辅酶A或乙酰乙酰-[³H]辅酶A缩合也产生标记的酶-S-HMG-SCoA。因此，乙酰辅酶A的乙酰部分以及乙酰乙酰辅酶A的乙酰乙酰和辅酶部分都被并入与酶共价连接的HMG-CoA中。在有利于预测的半胱氨酸-S-[¹⁴C]HMG-SCoA片段中分子内硫到氮酰基转移的条件下，对酶-S-[¹⁴C]HMG-SCoA进行蛋白水解消化。蛋白酶消化后的材料经过甲酸氧化产生N-[¹⁴C]HMG-半胱氨酸，表明HMG-CoA已通过活性位点半胱氨酸的-SH与酶共价结合。采用同位素捕获技术来测试乙酰-S-酶作为HMG-辅酶A合酶催化反应中间体的动力学活性。所提供的证据表明，乙酰乙酰辅酶A与乙酰-S-酶缩合形成酶-S-HMG-SCoA的速率比乙酰辅酶A对合酶的乙酰化作用或导致HMG-CoA的整个正向反应都要快。这些观察结果，连同酶-S-HMG-SCoA水解极其迅速的间接证据，表明合酶的乙酰化是HMG-CoA合成中的限速步骤。
