PMID:7893688
| Citation |
Shi, Y and Walsh, CT (1995) Active site mapping of Escherichia coli D-Ala-D-Ala ligase by structure-based mutagenesis. Biochemistry 34:2768-76 |
|---|---|
| Abstract |
Eleven Escherichia coli D-Ala-D-Ala ligase (DdlB) mutants, at K144, K215, and E270 in the ATP binding site, at E15, S150, H63, and R255 in the first D-Ala subsite, and at Y216, S281, L282, and D257 in the second D-Ala subsite, were constructed, purified, and examined for steady-state kinetic parameters, kcat and Kms for ATP, and both first (D-Ala1), and second (D-Ala2) D-alanines. Of these, E270Q, K215A, R255A, and D257N retained very low or no detectable activity consistent with X-ray structure based predictions for roles in Mg2+ coordination to beta, gamma-P of ATP (E270), coordination to transferring gamma-PO3 of ATP (K215), and coordination/orientation of nucleophilic COO- of D-Ala1 that attacks gamma-PO3 of ATP (R255, D257) and the side chain of R255, respectively. The substantial retention of activity in the Y216F mutant argues against the possibility that Y216 may be a catalytic base that deprotonates the alpha-NH3+ of D-Ala2 to attack the acyl phosphate form of D-Ala1. While all seven mutants that retain activities have a 300-2000 fold elevation in Km for D-Ala1 (1-2 microM in wild type), the S281A mutant has a 500-fold elevation in Km for D-Ala2, consistent with a proposed interaction with the COO- of D-Ala2. Similarly, the kinetics of inhibition by a slow-binding phosphinate inhibitor in the presence of ATP are most altered in the S281A mutant. |
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| Keywords |
Adenosine Diphosphate/metabolism; Adenosine Triphosphate/metabolism; Base Sequence; Binding Sites/genetics; Crystallography, X-Ray; DNA Primers/genetics; DNA, Bacterial/genetics; Dipeptides/chemistry; Dipeptides/metabolism; Escherichia coli/enzymology; Escherichia coli/genetics; Genes, Bacterial; Kinetics; Models, Molecular; Molecular Sequence Data; Molecular Structure; Mutagenesis, Site-Directed; Peptide Synthases/chemistry; Peptide Synthases/genetics; Peptide Synthases/metabolism; Protein Conformation |
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