Abstract
The structure of rat carboxypeptidase A2 (CPA2), which has a unique specificity for tryptophan-containing COOH-terminal peptides, has been determined in an unliganded state at 1.9-A resolution and refined to a crystallographic R-factor of 18.3%. Comparison of the structure of CPA2 with that of bovine carboxypeptidase A (referred to here as CPA1) reveals that the specificity of the former for larger amino acids probably arises from two amino acid replacements within the binding cavity (Thr268----Ala and Leu203----Met), coupled with differences in the positions of conserved residues in a surface loop on one face of the specificity pocket. The position of the reactive-site surface loop may be affected also by a disulfide bridge between Cys210 and Cys244. In this unliganded form of the enzyme, Tyr248 takes up a position interior to the specificity pocket and is distinct from that observed in bovine CPA1. The structural differences between CPA1 and CPA2 correlate strongly with crystallographically determined temperature factors and thus appear to be largest where the enzyme is flexible.
Highlights
From $the Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9050and the §Department of Biochemistryand Biophysics, University of California, San Francisco, California 94143
A crystallographic R-factor of 18.3%.Comparison of Purification-Rat carboxypeptidase A2 was purified from rat panthe structure of CPAZ with that of bovine carboxypep- creas (Pel-Freez, Rogers, AR) or from rat pancreas acetone powder tidase A reveals that the (Sigma) as described [1, 9, 31]
CPA2, CPAl, and carboxyspecificity of the former for larger amino acids prob- peptidase B were separated from each other, and from most of the ably arises from two amino acid replacements within other components of the acetone powder, by substrate affinity chrothe binding cavity (ThrZes+Alaand LeuZo3+Met)c, oupled with differences in the positions of conserved residues in a surface loop on one face of the specificity pocket
Summary
The PROLSQrefinement chemistry in the binding pocket are due to: 1) amino acid converged with a single model-building step at an overall R- replacements; 2) differences in the positions of conserved side factor of 22%;the resulting model differed from the XPLOP chains;and 3) differences inthe positions of main-chain refined model by a root mean square difference of 0.19 A. atoms. Correlation of the Structural Differences with B-factor and Amino Acid Replacement Frequency-As is apparent in Table 11,CPAB differs most from CPAl in the conformations of the loop structures, including the active site surface loop.
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