Substrate Specificity of Glycinamide Ribonucleotide Transformylase from Chicken Liver

Abstract

Several glycinamide ribonucleotide analogs have been prepared and evaluated as substrates and/or inhibitors of glycinamide ribonucleotide transformylase from chicken liver. The side chain modified analogs, in which the glycine side chain, R = CH2NH2, has been replaced by R = CH2NHCH3 and R = CH2CH2NH2, are substrates, with V/K (relative intensity) of 2.4% and 16.3%, respectively. Several carbocyclic analogs of glycinamide ribonucleotide, including the phosphonate derivative of carbocyclic glycinamide ribonucleotide, did not serve as substrates, but were inhibitors of the enzyme, competitive against glycinamide ribonucleotide, with Ki values ranging from 7.4 to 23.6 times the Km for glycinamide ribonucleotide. However, the O-phosphonate analog of carbocyclic glycinamide ribonucleotide did support enzymatic activity, with V/K (relative intensity) of 0.8%. In addition, glycinamide ribonucleoside was neither a substrate for, nor an inhibitor of, glycinamide ribonucleotide transformylase. Furthermore, alpha-glycinamide ribonucleotide had no effect on enzyme activity. These studies have begun to define the structural features of the nucleotide substrate required to support enzymatic activity.

Substrate Specificity of Glycinamide Ribonucleotide Synthetase from Chicken Liver

“…It also indicates that additional side chain length does not preclude enzymatic activity. These analogs also supported GAR transformylase activity (16).…”

“…GAR nucleoside was neither a substrate for nor an inhibitor of GAR synthetase, as was also observed with GAR transformylase (16). In contrast, phosphonates 3 and 12 showed different behavior with these two related activities.…”

“…1. The GAR analogs 1-5 and 12 were available from our study (16) of the substrate specificity of GAR transformylase, which catalyzes the next reaction in de novo purine biosynthesis. The synthesis of the carbocyclic PRA analogs 6 and 7 from a common chiral precursor (16) is illustrated in Scheme 1.…”

“…The results with the carbocycles again point to an important role for the ribose ring oxygen in substrate recognition. This contrasts with (Ϫ)-C-GAR utilization by GAR transformylase, the subsequent activity in de novo purine biosynthesis, which also uses GAR as a substrate, where removal of the ribose ring oxygen led to a small diminution in affinity (12,16). Comparison of the utilization of (Ϫ)-C-GAR (12) with that of its dideoxy (5) and its dideoxydidehydro (4) derivatives indicates that the cis-glycol does contribute to binding affinity, perhaps through hydrogen bond interactions.…”

“…The trifunctional enzyme was purified from chicken liver (15), and for kinetic studies, concentrated enzyme was dialyzed against 10 mM HEPES, pH 7.5, 20% glycerol. Analogs 1, 2, 4, and 5, ␣-GAR, ␤-GAR, and GAR nucleoside were prepared as described previously (16). The synthesis of phosphonates 3, 8, and 12 and related compounds will be reported elsewhere.…”