Reaction of brain hexokinase with a substrate-like reagent. Alkylation of a single thiol at the active site.
Reaction of brain hexokinase with a substrate-like reagent. Alkylation of a single thiol at the active site.
复制标题
脑己糖激酶与底物样试剂的反应。
作者：
G. Swarup;U. W. Kenkare
底物葡萄糖的类似物N-（溴乙酰基）-D-葡糖胺（GlcNBrAc）在pH 8.5和22 ℃下以假一级方式完全和不可逆地灭活牛脑线粒体己糖激酶。该试剂对己糖激酶的失活速率并不随试剂浓度的增加而线性增加，而是表现出明显的饱和效应，表明在失活步骤之前酶和试剂之间形成可逆的复合物。失活速率的pH依赖性表明，pKa = 9.1的酶上的基团被该试剂修饰。在pH 8.0时，该试剂的灭活速率非常慢，并且可以证明其是相对于底物葡萄糖的己糖激酶反应的竞争性抑制剂。底物葡萄糖和ATP强烈地保护酶免于失活反应。发现酶的失活伴随着两个巯基残基的烷基化，如通过形成约2 mol S-（羧甲基）-半胱氨酸/mol失活酶所示。用14 C标记的试剂处理酶导致约2 mol试剂/mol失活酶的掺入。然而，被葡萄糖保护的酶仍然显示出约1摩尔标记试剂/摩尔酶的掺入。从由该试剂灭活的酶的胰蛋白酶消化物中，获得两种标记的肽，如果在葡萄糖存在下进行标记反应，则其中一种不存在。这些结果表明，亲和试剂与两个硫醇反应，其中只有一个是至关重要的酶的活性，并位于其活性位点的区域。
An analogue of the substrate glucose, N-(bromoacetyl)-D-glucosamine (GlcNBrAc) inactivates bovine brain mitochondrial hexokinase completely and irreversibly in a pseudo-first-order fashion at pH 8.5 and 22 degrees C. The rate of inactivation of hexokinase by this reagent does not increase linearly with increasing reagent concentration but exhibits an apparent saturation effect, suggesting the formation of a reversible complex between the enzyme and the reagent prior to the inactivation step. The pH dependence of the rate of inactivation suggests that a group on the enzyme with pKa = 9.1 is being modified by this reagent. At pH 8.0 the rate of inactivation by this reagent is very slow, and it can be shown to be a competitive inhibitor of the hexokinase reaction with respect to the substrate glucose. The substrates glucose and ATP strongly protected the enzyme against the inactivation reaction. The inactivation of the enzyme was found to be accompanied by the alkylation of two sulfhydryl residues as shown by the formation of approximately 2 mol of S-(carboxymethyl)-cysteine/mol of inactivated enzyme. Treatment of the enzyme with 14C-labeled reagent results in the incorporation of approximately 2 mol of reagent/mol of inactivated enzyme. However, the enzyme protected by glucose still shows the incorporation of approximately 1 mol of the labeled reagent/mol of the enzyme. From a tryptic digest of the enzyme inactivated by this reagent, two labeled peptides were obtained, one of which was absent if the labeling reaction was carried out in presence of glucose. These results indicate that the affinity reagent reacts with two thiols, only one of which is crucial for the activity of the enzyme and is located in the region of its active site.