Dynamics of a mobile loop at the active site of Escherichia coli asparaginase

Asparaginase II from Escherichia coli is well-known member of the bacterial class II amidohydrolases. Enzymes of this family utilize a peculiar cataly tic mechanism in which a pair of threonine residues play pivotal roles. Ano ther common feature is a mobile surface loop that closes over the active si te when the substrates is bound. We have studied the motion of the loop by stopped-flow experiments using the fluorescence of tryptophan residues as t he spectroscopic probe. With wildtype enzyme the fluorescence of the only t ryptophan, W66, was monitored. Here asparagine induced a rapid closure of t he loop. The rate constants of the process (100-150 s(-1) at 4 degrees C) w ere considerably higher than those of the rate-limiting catalytic step. A m ore selective spectroscopic probe was generated by replacing W66 with tyros ine and Y25, a component of the loop, with tryptophan. In the resulting enz yme variant, k(cat) and the rate of loop movement were reduced by factors o f 10(2) and > 10(3), respectively, while substrate binding was unaffected. This indicates that the presence of tyrosine in position 25 is essential fo r both loop closure and catalysis. Numerical simulations of the observed tr ansients are consistent with a model where loop closure is an absolute prer equisite for substrate turnover. (C) 2000 Elsevier Science B.V. All rights reserved.