EVALUATION OF THE KINETIC MECHANISM OF ESCHERICHIA-COLI URIDINE DIPHOSPHATE-N-ACETYLMURAMATE-L-ALANINE LIGASE

Initial velocity methods were used to probe the kinetic mechanism of E scherichia coli uridine diphosphate-N-acetylmuramate:L-alanine ligase (UNAM:L-Ala ligase). When the activity (in the forward direction) vers us substrate concentration data were plotted in double-reciprocal form , all line patterns were intersecting The best fit of these data was t o the equation for an ordered mechanism with the following parameters: k(cat), 1000 +/- 100 min(-1); K-ma, 210 +/- 40 mu M; K-mb, 84 +/- 20 mu M; K-mc, 70 +/- 15 mu M; K-ia, 180 +/- 50 mu M; K-ib, 68 +/- 24 mu M. Initial velocity line patterns were also determined when the concen tration of one substrate was varied at different fixed concentrations of a second substrate while the third substrate was held at a concentr ation more than 100 times its K-m value. Reciprocal plots of data coll ected with either ATP or L-alanine present at more than 100 times thei r K-m values resulted in intersecting line patterns. Data collected wi th UNAM present at 100 times its K-m value gave a set of parallel line s. These data are consistent with UNAM binding as the second substrate in an ordered mechanism. ADP, uridine diphosphate-N-acetylmuramoyl-L- alanine (UNAMA), and phosphate were tested as product inhibitors versu s substrates. None of the products were competitive inhibitors versus L-alanine or UNAM, while the only observed competitive inhibition was ADP versus ATP. These results are consistent with an ordered kinetic m echanism wherein ATP binds first, UNAM binds second, and ADP is the la st product released. Rapid quench experiments were performed in the pr esence of all three substrates or in the presence of ATP and UNAM. The production of acid-labile phosphate as a function of time is characte rized by a burst phase followed by a slower linear phase with the rate close to k(cat) in the presence of all three substrates. Only a burst phase was observed for the time course of the reaction in the presenc e of ATP and UNAM. In both cases, the burst rate was identical. These observations are consistent with L-alanine being the third substrate t o bind in a sequential mechanism involving a putative acyl-phosphate i ntermediate.