Catalytic mechanism of a C-C hydrolase enzyme: evidence for a gem-diol intermediate, not an acyl enzyme.
Catalytic mechanism of a C-C hydrolase enzyme: evidence for a gem-diol intermediate, not an acyl enzyme.
复制标题
C-C水解酶的催化机制：偕二醇中间体而非酰基酶的证据。
作者：
S. M. Fleming;T. Robertson;G. Langley;T. Bugg
来自大肠杆菌的2-羟基-6-酮基-壬-2，4-二烯1，9-二酸5，6-水解酶（MhpC）催化苯丙酸分解代谢途径上的二醇外环裂变产物的水解裂解，并且是α/β水解酶家族的成员。该酶的催化机理先前已显示通过二烯醇底物的初始酮化进行（亨德森，I. M. J.，和Bugg，T. D. H.（1997）Biochemistry 36，12252-12258），然后进行立体特异性片段化。尽管在α/β水解酶家族中存在活性位点丝氨酸残基，但尝试使用（14）C-标记底物通过放射化学捕获方法验证推定的酰基酶中间体产生了<1%共价中间体的化学计量，这可以通过非酶过程来解释。相比之下，使用天然底物观察到5-6%的来自H（2）（18）O的两个原子的（18）O掺入琥珀酸中，这与偕二醇中间体的可逆形成一致。此外，在MhpC存在下，观察到（18）O从H（2）（18）O到不可水解的类似物4-酮基-壬-1，9-二酸的羰基中的时间依赖性掺入，这与酶催化的水对酮羰基的攻击一致。这些结果有利于涉及碱催化攻击水的催化机制，而不是活性位点丝氨酸的亲核攻击。这项工作的含义是，推定的活性位点丝氨酸在这种酶可能有一个替代功能，例如，作为一个基地。
2-Hydroxy-6-keto-nona-2,4-diene 1,9-dioic acid 5,6-hydrolase (MhpC) from Escherichia coli catalyses the hydrolytic cleavage of the extradiol ring fission product on the phenylpropionate catabolic pathway and is a member of the alpha/beta hydrolase family. The catalytic mechanism of this enzyme has previously been shown to proceed via initial ketonization of the dienol substrate (Henderson, I. M. J., and Bugg, T. D. H. (1997) Biochemistry 36, 12252-12258), followed by stereospecific fragmentation. Despite the implication of an active site serine residue in the alpha/beta hydrolase family, attempts to verify a putative acyl enzyme intermediate by radiochemical trapping methods using a (14)C-labeled substrate yielded a stoichiometry of <1% covalent intermediate, which could be accounted for by nonenzymatic processes. In contrast, incorporation of 5-6% of two atoms of (18)O from H(2)(18)O into succinic acid was observed using the natural substrate, consistent with the reversible formation of a gem-diol intermediate. Furthermore, time-dependent incorporation of (18)O from H(2)(18)O into the carbonyl group of a nonhydrolysable analogue 4-keto-nona-1,9-dioic acid was observed in the presence of MhpC, consistent with enzyme-catalyzed attack of water at the ketone carbonyl. These results favor a catalytic mechanism involving base-catalyzed attack of water, rather than nucleophilic attack of an active site serine. The implication of this work is that the putative active site serine in this enzyme may have an alternative function, for example, as a base.