Carboxypeptidase three-dimensional structure

Carboxypeptidase A was the first zinc enzyme to yield a three-dimensional structure to the X-ray crystallographic method, and the structure of an enzyme-pseudosubstrate complex provided a model for a precatalytic zinc-carbonyl interaction (Lipscomb etai, 1968). Comparative studies have been performed between carboxypeptidase A and thermolysin based on the results of X-ray crystallographic experiments (Argosetai, 1978 Kesterand Matthews, 1977 Monzingoand Matthews, 1984 Matthews, 1988 Christianson and Lipscomb, 1988b). Models of peptide-metal interaction have recently been utilized in studies of metal ion participation in hydrolysis (see e.g., Schepartz and Breslow, 1987). In these examples a dipole-ion interaction is achieved by virtue of a chelate interaction involving the labile carbonyl and some other Lewis base (e.g.. 

Thiosulfate cyanide sulfurtransferase symmetry in 78 TTiiouridine 234 Three-dimensional structures of aconitase 689 adenylate kinase 655 aldehyde oxido-reductase 891 D-amino acid oxidase 791 a-amylase, pancreatic 607 aspartate aminotransferase 57,135 catalytic intermediates 752 aspartate carbamyltransferase 348 aspartate chemoreceptor 562 bacteriophage P22 66 cadherin 408 calmodulin 317 carbonic acid anhydrase I 679 carboxypeptidase A 64 catalase 853 cholera toxin 333, 546 chymotrypsin 611 citrate synthase 702, 703 cutinase 134 cyclosporin 488 cytochrome c 847 cytochrome c peroxidase 849 dihydrofolate reductase 807 DNA 214, 223,228,229, 241 DNA complex... 

The considerable detail to which we now can understand enzyme catalysis is well illustrated by what is known about the action of carboxypeptidase A. This enzyme (Section 25-7B and Table 25-3) is one of the digestive enzymes of the pancreas that specifically hydrolyze peptide bonds at the C-terminal end. Both the amino-acid sequence and the three-dimensional structure of carboxypeptidase A are known. The enzyme is a single chain of 307 amino-acid residues. The chain has regions where it is associated as an a helix and others where it is associated as a /3-pIeated sheet. The prosthetic group is a zinc ion bound to three specific amino acids and one water molecule near the surface of the molecule. The amino acids bound to zinc are His 69, His 196, and Glu 72 the numbering refers to the position of the amino acid along the chain, with the amino acid at the /V-terminus being number l. The zinc ion is essential for the activity of the enzyme and is implicated, therefore, as part of the active site. 

Nilges, M., Gronenborn, A. M., Brunger, A. T. and Clore, G M. (1988). Determination of three-dimensional structures of proteins by simulated annealing with interproton distance restraints Application to crambin, potato carboxypeptidase inhibitor and barley serine proteinase inhibitor 2. Protein Eng. 2, 27-38. 

M. Nilges, A. M. Gronenborn, A. T. Brunger, and G. M. Clore, Protein Engin., 2,27 (1988). Determination of Three-Dimensional Structures of Proteins by Simulated Annealing with Interproton Distance Restraints. Application to Crambin, Potato Carboxypeptidase Inhibitor, and Barley Serine Proteinase Inhibitor 2. 

This discussion of the metalloexopeptidases has focused on the general role of these enzymes in the conversion of dietary proteins into amino acids. In particular, the apparent synergistic relationship which the pancreatic carboxypeptidases have with the major endopeptidases, trypsin, chymotrypsin, and pepsin, in order to facilitate formation of essential amino acids has been stressed. The chemical characteristics, metalloenzyme nature, and mechanistic details of a representative of each class of exopeptidase have been presented. Leucine aminopeptidase from bovine lens was shown to be subject to an unusual type of metal ion activation which may be representative of a more general situation. Carboxypeptidase A of bovine pancreas was discussed in terms of its three-dimensional structure, the implications of x-ray crystallography to mecha ... 

Zinc usually binds to proteins via residues of cysteine and histidine. Sometimes zinc is bound to residues of glutamate or aspartate. The zinc ion sometimes plays a catalytic role and sornetimes a structural role. In the latter case, it helps maintain the three-dimensional structure or conformation of the protein. For example carboxypeptidase A contains twr> atoms of zinc. One is required for catalytic activity and is bound to cysteine and histidine. The other, which plays a structural role, is bound only hr cysteine. Cytoplasmic supeioxide dismutase is a dimer, ft contains one atom of Cu and one of Zn + per subunit. The zinc is bound via three residues of histidine and one residue of aspartate. It is buried deep within the enzyme and serves a structural role. The copper atom is bound via four residues of histidine. It resides dose to the surface of the protein and participates in the chemistry of catalysis. 

Lipscomb, W. N. (1974) Relationship of the Three Dimensional Structure of Carboxypeptidase A to Catalysis. Possible Intermediates and pH Effects, Tetrahedron 30, 1725-1732. 

Bovine carboxypeptidase A was only the third protein, after myoglobin and lysozyme, to have its three-dimensional structure solved at high resolution. The active site zinc is bound to His-69, Glu-72, and His-196 (Figure 12.5), and to a water molecule. The zinc-bound water molecule is itself hydrogen bonded to Glu-270. Despite extensive experimental data, the mechanism of carboxypeptidase still remains controversial. Two major... 

Preliminary investigations involving a P-lactam-sensitive, bifunctional D-alanyl-carboxypeptidase—transpeptidase (C Pase—T Pase) from Streptomyces R61 have identified the three-dimensional structure and catalytic site of interaction with penicillins (63). 

The three-dimensional structure of many enzyme molecules, including hydrolytic enzymes such as lysozyme, chymotrypsin, ribonuclease, carboxypeptidase A, elastase, and papain, has been determined in recent years throu] the X-ray diffraction method, and the steric arrangement and function of amino acid residues at the active site has been elucidated (/). 

Fig. 2. Three-dimensional structural representations for zinc metall-oproteins. Comparison of the zinc ion-protein bonding interactions for zinc requiring enzymes (A—C) with the zinc-insulin hexamer (D, E). (A) Human carbonic anhydrase C, redrawn from Ref. (47) with permission. (B) Bovine carboxypeptidase Ay, redrawn from Ref. 30) with permission. (C) Bacillus thermoprotedyticus thermolysin, redrawn from Ref. 45) with permission. (D) and (E) Porcine Zn-insulin hexamer, taken from Ref. 48) with permission. The composite electron density maps in (D) and (E) show that each of the two zinc atoms present in the hexamer is within inner sphere bonding distance of three solvent molecules and three histidyl imidazolyl groups in an octahedral array about the metal ion. The position of one of the three equivalently positioned solvent molecules is indicated in (D). The electron density map in (E) shows the relative orientations of the three histidyl residues (His-BlO). (The atomic positions of one of the three equivalent histidyl groups are shown)...

Lipscomb 181) has presented a speculative discussion of the effects of pH on carboxypeptidase activity in relation to the high-resolution three-dimensional structures. In this interesting paper, Lipscomb proposes that the apparent pKa values observed in the pH profiles shown in Fig. 10 reflect respectively the ionization of Glu-270 (pKa 6.7) and the ionization of the zinc ion-coordinated water molecule (pKa 8.9). 

The human lysosmal serine carboxypeptidase CatA is a member of the a/p hydrolase enzyme family and therefore shares structural homology with yeast carboxypeptidase Y and cholinesterases [4] (Figure 23.1). The common feature shared by members of this family is a three-dimensional structure of eight p sheets connected by a-heUces resulting in a typical topological localization of the catalytic triad, in which only the His residue is conserved [2]. 

He used X-ray diffraction methods to determine the three-dimensional structure of proteins and then analyzed their function. Among the proteins studied by Lipscomb and coworkers were carboxypeptidase A, a digestive enzyme, and aspartate carbamoyltransferase, an enzyme from E. coli. 

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