A key role in catalysis for His89 of adenylosuccinate lyase of Bacillus subtilis.
Adenylosuccinate lyase of Bacillus subtilis is a tetrameric enzyme which catalyzes the cleavage of adenylosuccinate to AMP and fumarate. We have mutated His(89), one of three conserved histidines, to Gln, Ala, Glu, and Arg. The enzymes were expressed in Escherichia coli and purified to homogeneity. As compared to a specific activity of 1. 56 micromol of adenylosuccinate converted/min/mg protein for wild-type enzyme, the mutant enzymes exhibit specific activities of 0.0225, 0.0036, 0.0036, and 0.0009 for H89Q, H89A, H89E, and H89R, respectively. Circular dichroism and FPLC gel filtration reveal that mutant enzymes have a similar conformation and oligomeric state to that of wild-type enzyme. In H89Q, the K(M) for adenylosuccinate increases slightly to 2.5-fold that of wild-type, the K(M) for fumarate is elevated 3.3-fold, and the K(M) for AMP is 13 times higher than that observed in wild-type enzyme. The catalytic efficiency of the H89Q enzyme is compromised, with k(cat)/K(M) reduced 174-fold in the direction of AMP formation. These data suggest that His(89) plays a role in both the binding of the AMP portion of the substrate and in correctly orienting the substrate for catalysis. Incubation of H89Q with inactive H141Q enzyme [Lee, T. T., Worby, C., Bao, Z.-Q., Dixon, J. E., and Colman, R. F. (1999) Biochemistry 38, 22-32] leads to a 30-fold increase in activity. This intersubunit complementation indicates that His(89) and His(141) from different subunits participate in the active site and that both are required for catalysis.
Brosius
J L
JL
Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716, USA. rfcolman@udel.edu
Colman
R F
RF
D000264:Adenylosuccinate Lyase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D002384:Catalysis; D002942:Circular Dichroism; D004789:Enzyme Activation / Q000235:genetics; D006639:Histidine / Q000737:chemistry* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000138:chemical synthesis / Q000737:chemistry / Q000302:isolation & purification; D013696:Temperature
D011994:Recombinant Proteins; D006639:Histidine; D000264:Adenylosuccinate Lyase