Characterization of the dinuclear metal center of Pyrococcus furiosus prolidase by analysis of targeted mutants.
Prolidases are dipeptidases specific for cleavage of Xaa-Pro dipeptides. Pyrococcus furiosus prolidase is a homodimer having one Co-bound dinuclear metal cluster per monomer with one tightly bound Co(II) site and the other loosely bound (Kd 0.24 mM). To identify which Co site is tight-binding and which is loose-binding, site-directed mutagenesis was used to modify amino acid residues that participate in binding the Co1 (E-313 and H-284), the Co2 site (D-209) or the bidentate ligand (E-327). Metal-content, enzyme activity and CD-spectra analyses of D209A-, H284L-, and E327L-prolidase mutants show that Co1 is the tight-binding and Co2 the loose-binding metal center.
Du
Xuelian
X
Department of Microbiology, North Carolina State University, Box 7615, Raleigh, NC 27695, USA.
Tove
Sherry
S
Kast-Hutcheson
Karen
K
Grunden
Amy M
AM
D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics*; D001665:Binding Sites; D002384:Catalysis; D003035:Cobalt / Q000737:chemistry*; D004150:Dipeptidases / Q000737:chemistry* / Q000235:genetics*; D009154:Mutation; D020027:Pyrococcus furiosus / Q000201:enzymology*
D019843:Archaeal Proteins; D003035:Cobalt; D004150:Dipeptidases; C022449:proline dipeptidase