Ambiguities in mapping the active site of a conformationally dynamic enzyme by directed mutation. Role of dynamics in structure-function correlations in Escherichia coli adenylosuccinate synthetase.
On the basis of ligated crystal structures, Asn21, Asn38, Thr42, and Arg419 are not involved in the chemical mechanism of adenylosuccinate synthetase from Escherichia coli, yet these residues are well conserved across species. Purified mutants (Asp21 --> Ala, Asn38 --> Ala, Asn38 --> Asp, Asn38 --> Glu, Thr42 --> Ala, and Arg419 --> Leu) were studied by kinetics, circular dichroism spectroscopy, and equilibrium ultracentrifugation. Asp21 and Arg419 are not part of the active site, yet mutations at positions 21 and 419 lower kcat 20- and 10-fold, respectively. Thr42 interacts only through its backbone amide with the guanine nucleotide, yet its mutation to alanine significantly increases Km for all substrates. Asn38 hydrogen-bonds directly to the 5'-phosphoryl group of IMP, yet its mutation to alanine and glutamate has no effect on Km values, but reduces kcat by 100-fold. The mutation Asn38 --> Asp causes 10-57-fold increases in Km for all substrates along with a 30-fold decrease in kcat. At pH 5.6, however, the Asn38 --> Asp mutant is more active, yet binds IMP 100-fold more weakly, than the wild-type enzyme. Proposed mechanisms of ligand-induced conformational change and subunit aggregation can account for the properties of mutant enzymes reported here. The results underscore the difficulty of using directed mutations alone as a means of mapping the active site of an enzyme.
Wang
W
W
Department of Biochemistry and Biophysics, Iowa State University, Ames, Iowa 50011, USA.
Gorrell
A
A
Hou
Z
Z
Honzatko
R B
RB
Fromm
H J
HJ
D000265:Adenylosuccinate Synthase / Q000235:genetics*; D001665:Binding Sites; D002874:Chromosome Mapping; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D013329:Structure-Activity Relationship
D000265:Adenylosuccinate Synthase