| Abstract | Human glycinamide ribonucleotide transformylase (GART) (EC 2.1.2.2) is a validated target for 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 have been 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, N106Q and K170R, resulted in catalytically active enzymes, and these active mutant enzymes gave pH-rate profiles and a steady-state kinetic mechanism essentially identical to those of the native enzyme. All inactive mutants were able to bind both substrates, ruling out disrupted formation of the ternary complex as the source of inactivity. Differences between human and Escherichia coli GART, previously used as a model for the human enzyme, were evident. |
| Authors | Wanda Manieri, Molly E Moore, Matthew B Soellner, Pearl Tsang, Carol A Caperelli
(Affiliation: Division of Pharmaceutical Sciences, College of Pharmacy, University of Cincinnati Medical Center, P.O. Box 670004, Cincinnati, Ohio 45267, USA.)
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| Journal | Biochemistry
(Biochemistry)
Vol. 46
Issue 1
Pg. 156-63
(Jan 9 2007)
ISSN: 0006-2960 United States |
| PMID | 17198385
(Publication Type: Journal Article, Research Support, N.I.H., Extramural)
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| Chemical References |
- Recombinant Fusion Proteins
- Phosphoribosylglycinamide Formyltransferase
|
| Topics |
- Amino Acid Sequence
- Binding Sites
- Conserved Sequence
- Humans
- Hydrogen-Ion Concentration
- Kinetics
- Molecular Sequence Data
- Mutagenesis, Site-Directed
- Mutation
- Phosphoribosylglycinamide Formyltransferase
(chemistry, genetics, metabolism)
- Recombinant Fusion Proteins
(genetics, isolation & purification, metabolism)
- Substrate Specificity
|
