COMPLETE INACTIVATION OF ESCHERICHIA-COLI URIDINE PHOSPHORYLASE BY MODIFICATION OF ASP(5) WITH WOODWARDS REAGENT-K

Woodward's reagent K (WRK) completely inactivated Escherichia coli uri dine phosphorylase by reversible binding in the active site (K-i = 0.0 7 mM) with subsequent modification of a carboxyl (k(2) = 1.2 min(-1)). Neither substrate alone protected uridine phosphorylase from inactiva tion. The presence of phosphate did not affect the K-i and k(2) values . The addition of uracil or uridine led to a significant increase of b oth K-i (to 2.5 or 2.1 mM, respectively) and k(2) (to 6.1 or 4.8 min(- 1), respectively) values. Thus, WRK could react in accordance with slo w (high affinity) and fast (low affinity) mechanisms. Combined additio n of phosphate and uracil completely protected uridine phosphorylase. Tryptic digestion yielded a single modified peptide (4)-Asp(WRK)-Val-P he-His-Leu-Gly-Leu-Thr-Lys(13)). Treatment of the modified enzyme with hydroxylamine led to removal of the bulky WRK residue and replacement of the Asp(5) carboxyl by a hydroxamic group. The enzyme thus obtaine d recovered about 10% of initial specific activity, whereas its substr ate binding ability changed only moderately; the K-m values for phosph ate 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. Sc hwartz (1977) Eur. J. Biochem. 75, 217-224)) to 22.6 and 0.12 mM, resp ectively. The hydroxamic enzyme had higher thermostability than the na tive enzyme. The results obtained demonstrated the importance of the c arboxyl at position 5. The loss of activity after selective group repl acement is due to impaired stabilization of the transition state rathe r than to a decline in substrate affinity or change of the active site structure.