Human Glycinamide Ribonucleotide Transformylase:  Active Site Mutants as Mechanistic Probes

Abstract

Human glycinamide ribonucleotide transformylase (GART) (EC2.1.2.2) 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, D144, and the semi-conserved 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 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 E. coli GART, previously used as a model for the human enzyme, were evident.Glycinamide ribonucleotide transformylase (GART 1 , EC 2.1.2.2) catalyzes the transfer of the formyl group from N10-formyl tetrahydrofolate to the primary, side-chain amino group of glycinamide ribonucleotide (GAR) to yield formyl-glycinamide ribonucleotide (FGAR) and tetrahydrofolate (Scheme 1), ultimately resulting in the incorporation of C-8 into inosinic acid (IMP). This reaction is the third step and the first of two folate-dependent formyl transfers in the de novo purine biosynthetic pathway. GART was first discovered and partially characterized from pigeon liver in pioneering investigations by Warren and Buchanan (1).The critical role that purine nucleotides play as precursors to RNA and DNA led to the suggestion that inhibition of de novo purine biosynthesis might be a viable approach toward cancer chemotherapy (2-4). This suggestion was confirmed when it was demonstrated that 5,10-dideazatetrahydrofolate, a potent anti-tumor agent, has, as its mechanism of action, the inhibition of GART and, consequently, of de novo purine biosynthesis (5).This discovery led to a resurgence of interest in the de novo purine biosynthetic pathway and served as the impetus for numerous studies on the mechanism (6-9), structure (10-15), and † Supported by NIH grant GM61194. 1 ABBREVIATIONS: GART, glycinamide ribonucleotide transformylase; GAR, glycinamide ribonucleotide; FGAR, formylglycinamide ribonucleotide; GARS, glycinamide ribonucleotide synthetase; AIRS, aminoimidazole ribonucleotide synthetase; rhGART, recombinant human glycinamide ribonucleotide transformylase; fDDF, 10-formyl-5,8-dideazafolate; aDDF, 10-acetyl-5,8-dideazafolate; GAR-OH, hydroxyacetamide ribonucleotide. 23,24). In contrast, human GART comprises the C-terminal domain of a large (108 kDa), trifunctional enzyme that also catalyzes the synthesis of GAR (GARS) and the synthesis of aminoimidazole ribonucleotide (AIRS) (25,26). These additional activities catalyze steps 2 and 5 of the pathway. NIH Public AccessThe E. coli studies have provided useful information, including the identification of the wholly conserved residues, N106, H108...

Mutations in the Chinese hamster ovary cell GART gene of de novo purine synthesis

“…In this case, significant differences between the human GART protein and the E. coli protein have been observed (Manieri et al, 2007). These studies suggest that study of the human (or mammalian) GART and GARS proteins are important for their accurate characterization, although clearly comparisons of the mammalian and bacterial proteins will be useful, as described below.…”

“…The GART domain of the trifunctional GART gene has been cloned and expressed, allowing study of the GART monofunctional human protein. Structural and functional comparisons of the human and E. coli protein as well as targeted mutagenesis experiments have revealed important differences between these proteins (Manieri et al, 2007; Zhang et al, 2002). Similarly, structural and functional analysis coupled with targeted mutagenesis of the ADSL locus reveals important differences between the human, B. subtilis , and E. coli enzymes (Sivendran et al, 2004; Sivendran and Colman, 2008).…”

Protein preparation, crystallization and preliminary crystallographic studies ofBacillus subtilisglycinamide ribonucleotide transformylase

“…The crystal structures of Escherichia coli GART (Almassy et al, 1992;Chen et al, 1992;Klein et al, 1995;Greasley et al, 1999Greasley et al, , 2001, Mycobacterium tuberculosis GART (Zhang et al, 2009) and the human GART domain (Varney et al, 1997;Zhang et al, 2002Zhang et al, , 2003Dahms et al, 2005) have been determined. The catalytic mechanisms of GART have been studied in some detail (Caperelli & Giroux, 1997;Morikis et al, 2001;Su et al, 1998;Zhang et al, 2002;Manieri et al, 2007). GART is found as a single protein in prokaryotes, while in most eukaryotes it constitutes the C-terminal part of a monomeric but trifunctional protein with PurD-PurM-PurN activities (Almassy et al, 1992).…”