Complex between carboxypeptidase A and a hydrated ketomethylene substrate analogue.
The complex of carboxypeptidase A (CPA) with 5-amino-(N-t-butoxycarbonyl)-2-benzyl-4-oxo-6-phenylhexanoic acid (BBP), the ketomethylene substrate analogue of the peptide substrate N-(t-butoxycarbonyl)-L-phenylalanyl-L-phenylalanine, was studied by x-ray crystallographic methods. Interestingly, the enzyme specifically binds only one of four stereoisomers of BBP that were present in the buffer solution in which the CPA crystals were soaked. Furthermore, the species observed to bind to the enzyme is the hydrated form of the ketone. This is rather surprising since the hydrated form of BBP is expected to be present in aqueous solution at a concentration of less than 0.2%. Hence, the enzyme-inhibitor complex is most stable with a species resembling a structure along the reaction coordinate of a chemical reaction rather than a species resembling a reactant or a product. Important structural information regarding the catalytic conformations of active-site residues spanning the S'1-S2 subsites of the enzyme is provided from the results of these x-ray diffraction experiments. The structure of the CPA-hydrated BBP complex provides support for a promoted-water hydrolytic mechanism, although it is not certain whether the enzyme has actually participated in the hydration reaction at the ketone carbonyl of BBP.
Shoham
G
G
Department of Inorganic and Analytical Chemistry, Hebrew University, Israel.
Christianson
D W
DW
Oren
D A
DA
D001555:Benzene Derivatives; D002208:Caproates / Q000378:metabolism*; D002268:Carboxypeptidases / Q000378:metabolism*; D043422:Carboxypeptidases A; D007651:Keto Acids / Q000378:metabolism*; D011487:Protein Conformation; D014961:X-Ray Diffraction
D001555:Benzene Derivatives; D002208:Caproates; D007651:Keto Acids; C054766:5-amino-(N-t-butoxycarbonyl)-2-benzyl-4-oxo-6-phenylhexanoic acid; D002268:Carboxypeptidases; D043422:Carboxypeptidases A