The human glycinamide ribonucleotide transformylase domain: purification, characterization, and kinetic mechanism.
Glycinamide ribonucleotide transformylase catalyzes the third reaction of de novo purine biosynthesis, namely, the conversion of glycinamide ribonucleotide to N-formylglycinamide ribonucleotide, with concomitant conversion of 10-formyltetrahydrofolate to tetrahydrofolate. This activity has been shown to be a target for cancer chemotherapy, which has generated renewed interest in both the enzyme and the pathway. Moreover, in higher eukaryotes this activity constitutes the C-terminal domain of a monomeric protein which also catalyzes two additional reactions of de novo purine biosynthesis. In this study, the human glycinamide ribonucleotide transformylase domain has been expressed to high levels in Escherichia coli and purified to homogeneity. Our improved expression-purification system produces the desired activity exclusively in a soluble form and in higher abundance than previously achieved. The kinetic constants have been determined and the kinetic mechanism has been established as ordered-sequential, with the folate substrate binding first. The correspondence of these data to those obtained for the glycinamide ribonucleotide transformylase activity of the mammalian trifunctional enzyme indicates that the recombinant enzyme is fully functional.
Caperelli
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Division of Pharmaceutical Sciences, College of Pharmacy, University of Cincinnati Medical Center, Ohio 45267-0004, USA. Carol.Caperelli@UC.EDU
Giroux
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D000217:Acyltransferases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D019877:Hydroxymethyl and Formyl Transferases; D007700:Kinetics; D051026:Phosphoribosylglycinamide Formyltransferase; D011687:Purines / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
D004791:Enzyme Inhibitors; D011687:Purines; D011994:Recombinant Proteins; D019877:Hydroxymethyl and Formyl Transferases; D051026:Phosphoribosylglycinamide Formyltransferase; D000217:Acyltransferases