Glycinamide ribonucleotide synthase (EC 6.34.13)-aminoimidazole ribonucleotide synthase (EC 6.3.3.1) from Saccharomyces cerevisiae
- St. Petersburg State Univ. (Russian Federation); and others
Here we report for the first time the isolation and properties of GAR synthase (E2)-AIR synthase (E5) (E2-E5) from the yeast Saccharomyces cerevisiae. A vector containing the S. cerevisiae ADE5,7 gene was used to obtain a yeast strain accumulating ADE5,7 coded E2-E5 in amounts equivalent to {approximately}20% of the total soluble protein. The pH optimum for E2 activity in forward reaction is 7.5, in the reverse reaction 7.5 - 8.5; the E5 activity optimum is at pH 8.0. The temperature optimum for E2 is 42{degrees}C (in the reverse reaction), 35{degrees}C for E5. Data on the pH and temperature stability of E2-E5 are presented. The E2 K{sub m} values are ({mu}M): 49 {+-}4 for glycine, 64 {+-}5 for PRA, 144 {+-} 10 for ATP, 21 {+-} 2 for GAR, 7 {+-} 1 for ADP, and 320 {+-} 30 for P{sub i}. The E5 K{sub m} values are ({mu}M): 80 {+-} 9 for GAM and 500 {+-} 52 for ATP. E2 and E5 are inactive in the absence of Mg{sup 2+}. E5 is K{sup +} -dependent. 2{prime}-dATP, GTP, ITP, UTP, and CTP are not substrates for E5, while AMP is a competitive E5 inhibitor relative to ATP (with K{sub i} = 700 {+-} 80 {mu}M). Hence, regulation of purine biosynthesis by one of its end products in S. cerevisiae occurs at the fifth stage of biosynthesis, supplementing the inhibition of the first enzyme. The molecular mass of the E2-E5 subunits is {approximately}87 kD. The native form is dimeric: the molecular masses of the enzyme determined by three different methods are 170, 175, and 200 kD. Short-term chymotrypsin treatment of E2-E5 cleaves the polypeptide chain, presumably in an interdomain region; as a result, E5 activity disappears while E2 activity is retained. 45 refs., 6 figs., 2 tabs.
- OSTI ID:
- 433231
- Journal Information:
- Biochemistry (Eaton), Vol. 60, Issue 12; Other Information: PBD: Dec 1995
- Country of Publication:
- United States
- Language:
- English
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