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. 1981 Sep;78(9):5455–5459. doi: 10.1073/pnas.78.9.5455

Binding of ligands to the active site of carboxypeptidase A.

D C Rees, W N Lipscomb
PMCID: PMC348764  PMID: 6946483

Abstract

We compare the detailed binding modes of the 39-amino acid inhibitor from potatoes, glycyl-L-tyrosine, the ester analogue CH3OC6H4(CO)CH2CH(CO2(-))C6H5, and indole acetate to the exopeptidase carboxypeptidase A (EC 3.4.17.1). In the potato inhibitor, cleavage of the COOH-terminal glycine-39 leaves a new carboxylate anion of valine-38 having one oxygen on zinc and the other as a receptor of a hydrogen bond from tyrosine-248 of carboxypeptidase. Tyrosine-248 also receives a hydrogen bond from the amide proton of the originally penultimate peptide bond between tyrosine-37 and valine-38. This hydrogen bond suggests product stabilization which is available to peptides and depsipeptides but not to esters lacking an equivalent peptide bond (nonspecific esters). Also, this structure may represent the intermediate binding step for the uncleaved substrate as it moves along the binding subsites. In particular, this may be the binding mode for the substrate after association of the COOH-terminal region of the substrate with the residues at binding subsite S2 (tyrosine-198, phenylalanine-279, and arginine-71) and preceding entry into the catalytic site S1'. These stabilized complexes allow some understanding of the effect of indole acetate, shown here to bind in the pocket at S1', as a competitive inhibitor for esters (for which entry into S1' precedes the rate-determining catalytic step for hydrolysis) and as a noncompetitive inhibitor for peptides (for which entry into S1' is rate limiting). These results, including the binding mode of the ester analogue, are consistent with the original proposal from x-ray studies that both esters and peptides are cleaved with the carboxy terminus at S1', although not necessarily by the same chemical steps.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Abramowitz N., Schechter I., Berger A. On the size of the active site in proteases. II. Carboxypeptidase-A. Biochem Biophys Res Commun. 1967 Dec 29;29(6):862–867. doi: 10.1016/0006-291x(67)90299-9. [DOI] [PubMed] [Google Scholar]
  2. Auld D. S., Holmquist B. Carboxypeptidase A. Differences in the mechanisms of ester and peptide hydrolysis. Biochemistry. 1974 Oct 8;13(21):4355–4361. doi: 10.1021/bi00718a018. [DOI] [PubMed] [Google Scholar]
  3. Cleland W. W. Determining the chemical mechanisms of enzyme-catalyzed reactions by kinetic studies. Adv Enzymol Relat Areas Mol Biol. 1977;45:273–387. doi: 10.1002/9780470122907.ch4. [DOI] [PubMed] [Google Scholar]
  4. Harrison L. W., Auld D. S., Vallee B. L. Intramolecular arsanilazotyrosine-248-Zn complex of carboxypeptidase A: a monitor of multiple conformational states in solution. Proc Natl Acad Sci U S A. 1975 Nov;72(11):4356–4360. doi: 10.1073/pnas.72.11.4356. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Hass G. M., Hermodson M. A. Amino acid sequence of a carboxypeptidase inhibitor from tomato fruit. Biochemistry. 1981 Apr 14;20(8):2256–2260. doi: 10.1021/bi00511a029. [DOI] [PubMed] [Google Scholar]
  6. Johansen J. T., Vallee B. L. Conformations of arsanilazotyrosine-248 carboxypeptidase A alpha, beta, gamma, comparison of crystals and solution. Proc Natl Acad Sci U S A. 1973 Jul;70(7):2006–2010. doi: 10.1073/pnas.70.7.2006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Johansen J. T., Vallee B. L. Differences between the conformation of arsanilazotyrosine 248 of carboxypeptidase A in the crystalline state and in solution. Proc Natl Acad Sci U S A. 1971 Oct;68(10):2532–2535. doi: 10.1073/pnas.68.10.2532. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Johansen J. T., Vallee B. L. Environment and conformation dependent sensitivity of the arsanilazotyrosine-248 carboxypeptidase A chromophore. Biochemistry. 1975 Feb 25;14(4):649–660. doi: 10.1021/bi00675a001. [DOI] [PubMed] [Google Scholar]
  9. Lipscomb W. N. Carboxypeptidase A mechanisms. Proc Natl Acad Sci U S A. 1980 Jul;77(7):3875–3878. doi: 10.1073/pnas.77.7.3875. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Lipscomb W. N., Hartsuck J. A., Reeke G. N., Jr, Quiocho F. A., Bethge P. H., Ludwig M. L., Steitz T. A., Muirhead H., Coppola J. C. The structure of carboxypeptidase A. VII. The 2.0-angstrom resolution studies of the enzyme and of its complex with glycyltyrosine, and mechanistic deductions. Brookhaven Symp Biol. 1968 Jun;21(1):24–90. [PubMed] [Google Scholar]
  11. Quiocho F. A., McMurray C. H., Lipscomb W. N. Similarities between the conformation of arsanilazotyrosine 248 of carboxypeptidase A in the crystalline state and in solution. Proc Natl Acad Sci U S A. 1972 Oct;69(10):2850–2854. doi: 10.1073/pnas.69.10.2850. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Rees D. C., Honzatko R. B., Lipscomb W. N. Structure of an actively exchanging complex between carboxypeptidase A and a substrate analogue. Proc Natl Acad Sci U S A. 1980 Jun;77(6):3288–3291. doi: 10.1073/pnas.77.6.3288. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Rees D. C., Lewis M., Honzatko R. B., Lipscomb W. N., Hardman K. D. Zinc environment and cis peptide bonds in carboxypeptidase A at 1.75-A resolution. Proc Natl Acad Sci U S A. 1981 Jun;78(6):3408–3412. doi: 10.1073/pnas.78.6.3408. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Rees D. C., Lipscomb W. N. Structure of the potato inhibitor complex of carboxypeptidase A at 2.5-A resolution. Proc Natl Acad Sci U S A. 1980 Aug;77(8):4633–4637. doi: 10.1073/pnas.77.8.4633. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Scheule R. K., Van Wart H. E., Vallee B. L., Scheraga H. A. Resonance Raman spectroscopy of arsanilazocarboxypeptidase A: conformational equilibria in solution and crystal phases. Biochemistry. 1980 Feb 19;19(4):759–766. doi: 10.1021/bi00545a023. [DOI] [PubMed] [Google Scholar]
  16. Spilburg C. A., Bethune J. L., Valee B. L. Kinetic properties of crystalline enzymes. Carboxypeptidase A. Biochemistry. 1977 Mar 22;16(6):1142–1150. doi: 10.1021/bi00625a018. [DOI] [PubMed] [Google Scholar]
  17. Vallee B. L., Riordan J. F., Bethune J. L., Coombs T. L., Auld D. S., Sokolovsky M. A model for substrate binding and kinetics of carboxypeptidase A. Biochemistry. 1968 Oct;7(10):3547–3556. doi: 10.1021/bi00850a032. [DOI] [PubMed] [Google Scholar]

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