Substrate Specificity of Glycinamide Ribonucleotide Synthetase from Chicken Liver

Abstract

Several analogs of glycinamide ribonucleotide and phosphoribosylamine have been prepared and evaluated as substrates for glycinamide ribonucleotide synthetase purified from chicken liver. Glycinamide ribonucleotide analogs include side chain modifications wherein the glycine side chain (R ‫؍‬ CH 2 NH 2 ) has been replaced by R ‫؍‬ CH 2 NHCH 3 and R ‫؍‬ CH 2 CH 2 NH 2 , ribose ring replacement by chiral cyclopentane and cyclopentene derivatives, and phosphate replacement by phosphonates. All of these, with the exception of the O-phosphonate, served as substrates for the reverse enzymatic reaction, with V max values comparable to that obtained with glycinamide ribonucleotide, although the K m values ranged from 21 to 118 times the K m for glycinamide ribonucleotide. Analogs of phosphoribosylamine examined as substrates for the forward reaction consist of chiral derivatives of cyclopentane and cyclopentene and a chiral carbocyclic phosphonate. These also served as substrates, with K m values ranging from 5 to 23 times the K m for phosphoribosylamine and with diminished V max values. These studies have begun to define the structural features of the nucleotide substrate necessary to support enzymatic activity. Sarcosine (N-methylglycine) and ␤-alanine were also accepted as substrates, albeit with reduced affinity compared with glycine.

Human SHMT inhibitors reveal defective glycine import as a targetable metabolic vulnerability of diffuse large B-cell lymphoma

“…Collectively, these results suggest a hierarchy in the sensitivity of different intracellular metabolic products to glycine levels: glutathione synthesis is most sensitive, followed by purine synthesis, with protein synthesis most resistant. This hierarchy is consistent with biochemical measurements of the K m values of the relevant enzymes: the glycyl-tRNA amino acid synthase has a lower K m for glycine (15 μM) than that found in glycinamide ribonucleotide synthetase (45 μM) or glutathione synthetase (452 μM) (26)(27)(28).…”

Proteomics reveals changes in hepatic proteins during chicken embryonic development: an alternative model to study human obesity

“…A recent study reported that NT5C overexpression decreased dNTP pools and negatively regulated nucleotide synthesis [ 50 ]. PPAT is a key enzyme in the first reaction of de novo purine biosynthesis [ 51 ] and XDH is involved in the oxidative metabolism of purines [ 52 ]. At the end of incubation, the beak of chicken embryo is unchanged and hatching muscle development matures.…”

A New View into the Regulation of Purine Metabolism: The Purinosome

“…The rationale for compartmentalization has largely relied on kinetic arguments. Specifically, the product of the first reaction in the de novo purine biosynthetic pathway, PRA, has a very short solution half-life and may directly transfer to the subsequent enzyme, GART, through complexation of the two enzymes [4, 31, 32]. Similarly, GART catalyzes non-concerted steps within the same pathway (Figure 1) and requires the activity of FGAMS for the fourth step raising the possibility that FGAMS interacts with GART [33].…”