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Kinetic and crystallographic studies of Escherichia coli UDP‐ N ‐acetylmuramate:L‐alanine ligase
Author(s) -
Emanuele John J.,
Jin Haiyong,
Jacobson Bruce L.,
Chang Chiehying Y.,
Einspahr Howard M.,
Villafranca Joseph J.
Publication year - 1996
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.5560051219
Subject(s) - chemistry , dna ligase , peptidoglycan , enzyme , stereochemistry , tris , crystallography , biochemistry
Abstract Uridine diphosphate‐ N ‐acetylmuramate:L‐alanine ligase (EC 6.3.2.8, UNAM:L‐Ala ligase or MurC gene product) catalyzes the ATP‐dependent ligation of the first amino acid to the sugar moiety of the peptidoglycan precursor. This is an essential step in cell wall biosynthesis for both gram‐positive and gram‐negative bacteria. Optimal assay conditions for initial velocity studies have been established. Steady‐state assays were carried out to determine the effect of various parameters on enzyme activity. Factors studied included: cation specificity, ionic strength, buffer composition and pH. At 37 °C and pH 8.0, k cat was equal to 980 40 min −1 , while K m values for ATP, UNAM, and L‐alanine were, 130 10, 44 3, and 48 6 μM, respectively. Of the metals tested only Mn, Mg, and Co were able to support activity. Sodium chloride, potassium chloride, ammonium chloride, and ammonium sulfate had no effect on activity up to 75 mM levels. The enzyme, in appropriate 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 produced two crystal forms. One of these has been characterized by X‐ray diffraction as monoclinic, space group C2, with cell dimensions a = 189.6, b = 92.1, c = 75.2 Å, β = 105°, and two 54 kDa molecules per asymmetric unit. It was discovered that the enzyme will hydrolyze ATP in the absence of L‐alanine. This L‐alanine independent activity is dependent upon the concentrations of both ATP and UNAM; k cat for this activity is less than 4% of the biosynthetic activity measured in the presence of saturating levels of L‐alanine. Numerous L‐alanine analogs tested were shown to stimulate ATP hydrolysis. A number of these L‐alanine analogs produced novel products as accessed by HPLC and mass spectral analysis. All of the L‐alanine analogs tested as inhibitors were competitive versus L‐alanine.