KINETIC AND CRYSTALLOGRAPHIC STUDIES OF ESCHERICHIA-COLI UDP-N-ACETYLMURAMATE-L-ALANINE LIGASE

Uridine diphosphate-N-acetylmuramate:L-alanine ligase (EC 6.3.2.8, UNA M:L-Ala ligase or MurC gene product) catalyzes the ATP-dependent ligat ion of the first amino acid to the sugar moiety of the peptidoglycan p recursor. This is an essential step in cell wall biosynthesis for both gram-positive and gram-negative bacteria. Optimal assay conditions fo r initial velocity studies have been established. Steady-state assays were carried out to determine the effect of various parameters on enzy me activity. Factors studied included: cation specificity, ionic stren gth, buffer composition and pH. At 37 degrees C and pH 8.0, k(cat) was equal to 980 +/- 40 min(-1), while K-m values for ATP, UNAM, and L-al anine were, 130 +/- 10, 44 +/- 3, and 48 +/- 6 mu M, respectively. Of the metals tested only Mn, Mg, and Co were able to support activity. S odium chloride, potassium chloride, ammonium chloride, and ammonium su lfate had no effect on activity up to 75 mM levels. The enzyme, in app ropriate buffer, was stable enough to be assayed over the pH range of 5.6 to 10.1, pH profiles of V-max/K-m for the three substrates and of V-max were obtained. Crystallization experiments with the enzyme produ ced two crystal forms. One of these has been characterized by X-ray di ffraction as monoclinic, space group C2, with cell dimensions a = 189. 6, b = 92.1, c = 75.2 Angstrom, beta = 105 degrees, and two 54 kDa mol ecules per asymmetric unit. It was discovered that the enzyme will hyd rolyze ATP in the absence of L-alanine. This L-alanine independent act ivity is dependent upon the concentrations of both ATP and UNAM; k(cat ) for this activity is less than 4% of the biosynthetic activity measu red in the presence of saturating levels of L-alanine. Numerous L-alan ine analogs tested were shown to stimulate ATP hydrolysis. A number of these L-alanine analogs produced novel products as accessed by HPLC a nd mass spectral analysis. All of the L-alanine analogs tested as inhi bitors were competitive versus L-alanine.