title: Dynamics of a mobile loop at the active site of Escherichia coli asparaginase.
abstract: Asparaginase II from Escherichia coli is well-known member of the bacterial class II amidohydrolases. Enzymes of this family utilize a peculiar catalytic mechanism in which a pair of threonine residues play pivotal roles. Another common feature is a mobile surface loop that closes over the active site when the substrates is bound. We have studied the motion of the loop by stopped-flow experiments using the fluorescence of tryptophan residues as the spectroscopic probe. With wild-type enzyme the fluorescence of the only tryptophan, W66, was monitored. Here asparagine induced a rapid closure of the loop. The rate constants of the process (100-150 s(-1) at 4 degrees C) were considerably higher than those of the rate-limiting catalytic step. A more selective spectroscopic probe was generated by replacing W66 with tyrosine and Y25, a component of the loop, with tryptophan. In the resulting enzyme variant, k(cat) and the rate of loop movement were reduced by factors of 10(2) and >10(3), respectively, while substrate binding was unaffected. This indicates that the presence of tyrosine in position 25 is essential for both loop closure and catalysis. Numerical simulations of the observed transients are consistent with a model where loop closure is an absolute prerequisite for substrate turnover.
authors.affiliation: Chemistry Department, University of Yangon, Myanmar.
authors.forename: H P
authors.initials: HP
authors.lastname: Aung
authors.forename: M
authors.initials: M
authors.lastname: Bocola
authors.forename: S
authors.initials: S
authors.lastname: Schleper
authors.forename: K H
authors.initials: KH
authors.lastname: Röhm
mesh_terms: D001215:Asparaginase / Q000737:chemistry / Q000378:metabolism*; D001216:Asparagine / Q000737:chemistry; D001665:Binding Sites; D002384:Catalysis; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005453:Fluorescence; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D013696:Temperature; D013997:Time Factors; D014364:Tryptophan / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry
chemical_list: D014443:Tyrosine; D001216:Asparagine; D014364:Tryptophan; D001215:Asparaginase