The structure of carboxypeptidase A. VIII. Atomic interpretation at 0.2 nm resolution, a new study of the complex of glycyl-L-tyrosine with CPA, and mechanistic deductions

Abstract

Bovine pancreatic carboxypeptidase Aa (CPA),J the subject of these studies, is a zinc containing enzyme of molecular weight 34 600, which catalyses the hydrolysis of polypeptides and esters at the C-terminal peptide or ester bond. Experiments to date have shown that in order to be hydrolysed, the substrate must contain a C-terminal residue in the l configuration, with the carboxyl group free and α to the peptide or ester bond which is to be cleaved. In addition, the reaction is favoured if the C-terminal residue is aromatic. Our crystallographic studies of CPA have yielded electron density maps of the native enzyme at 0.6 nm (Lipscomb, Coppola, Hartsuck, Ludwig, Muirhead, Searl & Steitz 1966), 0.28 nm (Ludwig, Hartsuck, Steitz, Muirhead, Coppola, Reeke & Lipscomb 1967) and 0.20 nm resolution (Reeke, Hartsuck, Ludwig, Quiocho, Steitz & Lipscomb 1967; Lipscomb, Hartsuck, Reeke, Quiocho, Bethge, Ludwig, Steitz, Muirhead & Coppola 1968). Concurrently, a study of the binding of a number of substrates and inhibitors at 0.6 nm resolution was under way (Steitz, Ludwig, Quiocho & Lipscomb 1967). Subsequently, the most promising of these complexes, that of glycyl-L-tyrosine with CPA, was carried to atomic resolution (Reeke et al. 1967; Lipscomb et al. 1968). Even though chemical sequence is not available for several of the binding and catalytic groups of the enzyme, we have been able to deduce the identity of the binding residue Arg-145 and the catalytic residue Glu-270 (Reeke et al. 1967), to describe the mode of binding of Gly-Tyr, to extrapolate these conclusions to the binding of polypeptides, and to propose a mechanism, with certain ambiguities, for the action of the enzyme (Lipscomb et al. 1968). We have now completed the detailed atomic interpretation of the 0.20 nm electron density map, supplying residue identifications where the primary sequence is not available, and have subjected the resulting atomic coordinates to a model building procedure (Diamond 1966) which forces them to conform to standard bond distances and angles. The improved coordinates have been entered in a structure factor calculation, which gave a standard crystallographic R factor of 0.44.

Crystal Structure of Carboxypeptidase A Complexed with d-Cysteine at 1.75 Å − Inhibitor-Induced Conformational Changes,

“…In structures of CPD bound to transition-state analogue inhibitors, Tyr248 is observed to swivel by 12 Å to donate a hydrogen bond to the inhibitor carboxylate. The scale of this movement was presented as evidence in support of the "induced fit" hypothesis of enzyme action (50,51). However, there is an orthorhombic crystal form of native CPD in which Tyr248 is positioned inside the active site similar to its conformation when a substrate is bound (52).…”

A Mechanism of Action for Carboxypeptidase A

“…The general random pathway model proposed is in harmony with crystallographic studies by Lipscomb et al (3). It is also able to offer a resolution to the disparity between structural data and previous interpretations of, and models for, substrate kinetic data.…”

“…The presence of rearrangement steps is deemed feasible by the fact the Lipscomb et al (3) noted that the native enzyme undergoes several'conformational changes with the binding of Gly-Tyr. With relaxation spectrometry, Hammes (17,18) found evidence to support the hypothesis that rapid conformational changes or isomerizations involving binary and ternary complexes occur in dehydrogenase systems, and evidence for the existence of at least eight different states of ribonuclease A.…”

“…The zinc atom plays an integral role in the cleavage of the substrate; it polarizes the carbonyl group of the-substrate, Zn+. ..0O--Cs+, in order to render the carbon atom of this carbonyl group more susceptible to nucleophilic attack (3). 2970…”

The Physical State Dependence of Carboxypeptidase A α and A γ Kinetics

“…In spite of existent data demonstrating differences in the rates at which crystals and solutions hydrolyze CbzGly-Phe and the questions raised regarding the structural significance of these differences (15), substrate model building experiments were performed based on solution kinetics (9,23). Carboxypeptidasesubstrate complexes did not prove susceptible to direct crystallographic analysis; hence, such simulations with substrate models were substituted to localize substratebinding sites to account for their catalytic, activating, and inhibiting modes (9,23).…”