GALACTOSE-1-PHOSPHATE URIDYLYLTRANSFERASE - IDENTIFICATION OF HISTIDINE-164 AND HISTIDINE-166 AS CRITICAL RESIDUES BY SITE-DIRECTED MUTAGENESIS
GALACTOSE-1-PHOSPHATE URIDYLYLTRANSFERASE - IDENTIFICATION OF HISTIDINE-164 AND HISTIDINE-166 AS CRITICAL RESIDUES BY SITE-DIRECTED MUTAGENESIS
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DOI：
10.1021/bi00431a019
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发表时间：
1989-03-07
期刊：
影响因子：
2.9
通讯作者：
FREY, PA
中科院分区：
文献类型：
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作者：
FIELD, TL;REZNIKOFF, WS;FREY, PA
半乳糖-1-磷酸尿苷基转移酶通过双位移机制催化udp -葡萄糖和半乳糖-1-p与udp -半乳糖和葡萄糖-1-p的相互转化，该机制涉及尿苷基酶中组氨酸残基的N3位置。对大肠杆菌中含有的编码尿苷基转移酶的galT基因进行了测序，并根据推导出的该蛋白氨基酸序列确定了15个组氨酸残基的位置。已经产生了15个突变基因，每个突变基因中15个组氨酸密码子中的一个被改变为天冬酰胺密码子，并用于改造大肠杆菌菌株JM101。当对转化子的提取物进行尿苷基转移酶检测时，13个表现出高水平的活性。两种含有突变型尿苷基转移酶的提取物表现出低于对照水平的活性。这些突变蛋白H164N和H166N被过表达、分离，并测试了它们形成强制尿苷基酶中间体的能力。H164N和H166N突变蛋白都不能形成中间产物。因此，His-164和His-166对活性都是至关重要的，它们的接近表明它们都在活性位点。一种是必不可少的亲核催化剂，其中尿酰基在中间体中与之结合，另一种具有同样重要但尚不清楚的功能。活性位点序列His(164)-Pro-His（166）在大肠杆菌、人类、酵母菌和链霉菌中保守。
Galactose-1-phosphate uridylyltransferase catalyzes the interconversion of UDP-glucose and galactose-1-P with UDP-galactose and glucose-1-P by a double-displacement mechanism involving the N3 position of a histidine residue in the uridylyl enzyme. The galT gene of Escherichia coli, which codes for the uridylyltransferase and is contained in a plasmid for transformation of E. coli, has been sequenced, and the positions of the 15 histidine residues have been determined from the deduced amino acid sequence of this protein. Fifteen mutant genes, in each of which one of the 15 histidine codons has been changed to an asparagine codon, have been generated and used to transform the E. coli strain JM101. When extracts of the transformants were assayed for uridylyltransferase, 13 exhibited high levels of activity. Two of the extracts containing mutant uridylyltransferase exhibited less than control levels of activity. These mutant proteins, H164N and H166N, were overexpressed, isolated, and tested for their ability to form the compulsory uridylyl enzyme intermediate. Neither the H164N nor the H166N mutant proteins could form the intermediate. Thus, both His-164 and His-166 are critical for activity, and their proximity suggests that both are in the active site. One is the essential nucleophilic catalyst to which the uridylyl group is bonded in the intermediate, and the other serves an equally important, as yet unknown, function. The active-site sequence His(164)-Pro-His(166) is conserved in this enzymes from E. coli, humans, Saccharomyces, and Streptomyces.