Complete inactivation of Escherichia coli uridine phosphorylase by modification of Asp5 with Woodward's reagent K.
Woodward's reagent K (WRK) completely inactivated Escherichia coli uridine phosphorylase by reversible binding in the active site (Ki = 0.07 mM) with subsequent modification of a carboxyl (k2 = 1.2 min-1). Neither substrate alone protected uridine phosphorylase from inactivation. The presence of phosphate did not affect the Ki and k2 values. The addition of uracil or uridine led to a significant increase of both Ki (to 2.5 or 2.1 mM, respectively) and k2 (to 6.1 or 4.8 min-1, respectively) values. Thus, WRK could react in accordance with slow (high affinity) and fast (low affinity) mechanisms. Combined addition of phosphate and uracil completely protected uridine phosphorylase. Tryptic digestion yielded a single modified peptide (Ser4-Asp(WRK)-Val-Phe-His-Leu-Gly-Leu-Thr-Lys13). Treatment of the modified enzyme with hydroxylamine led to removal of the bulky WRK residue and replacement of the Asp5 carboxyl by a hydroxamic group. The enzyme thus obtained recovered about 10% of initial specific activity, whereas its substrate binding ability changed only moderately; the Km values for phosphate and uridine were changed from 5.1 and 0.19 mM (or 7.3 and 0.14 mM according to Leer et al. (Leer, J.C., Hammer-Jespersen, K., and M. Schwartz (1977) Eur. J. Biochem. 75, 217-224)) to 22.6 and 0.12 mM, respectively. The hydroxamic enzyme had higher thermostability than the native enzyme. The results obtained demonstrated the importance of the carboxyl at position 5. The loss of activity after selective group replacement is due to impaired stabilization of the transition state rather than to a decline in substrate affinity or change of the active site structure.
Komissarov
A A
AA
State Scientific Centre of Russian Federation GNII Genetika, Moscow.
Romanova
D V
DV
Debabov
V G
VG
D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000737:chemistry*; D001665:Binding Sites; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology*; D006877:Hydroxamic Acids / Q000737:chemistry; D007202:Indicators and Reagents; D007555:Isoxazoles / Q000737:chemistry*; D007700:Kinetics; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D014543:Uridine Phosphorylase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000302:isolation & purification
D006877:Hydroxamic Acids; D007202:Indicators and Reagents; D007555:Isoxazoles; D001224:Aspartic Acid; C009686:N-ethyl-5-phenylisoxazolium-3'-sulfonate; D014543:Uridine Phosphorylase