Human Glycinamide Ribonucleotide Transformylase: Active Site Mutants as Mechanistic Probes
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- 作者：Wanda Manieri ; Molly E. Moore ; Matthew B. Soellner ; Pearl Tsang ; Carol A. Caperelli
- 刊名：Biochemistry
- 出版年：2007
- 出版时间：January 9, 2007
- 年：2007
- 卷：46
- 期：1
- 页码：156 - 163
- 全文大小：91K
- 年卷期：v.46,no.1(January 9, 2007)
- ISSN：1520-4995
文摘
Human glycinamide ribonucleotide transformylase (GART) (EC 2.1.2.2) is a validated targetfor cancer chemotherapy, but mechanistic studies of this therapeutically important enzyme are limited.Site-directed mutagenesis, initial velocity studies, pH-rate studies, and substrate binding studies havebeen employed to probe the role of the strictly conserved active site residues, N106, H108, and D144,and the semiconserved K170 in substrate binding and catalysis. Only two conservative substitutions, N106Qand K170R, resulted in catalytically active enzymes, and these active mutant enzymes gave pH-rateprofiles and a steady-state kinetic mechanism essentially identical to those of the native enzyme. Allinactive mutants were able to bind both substrates, ruling out disrupted formation of the ternary complexas the source of inactivity. Differences between human and Escherichia coli GART, previously used asa model for the human enzyme, were evident.