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Refined structure of bovine carboxypeptidase A at 1.25 A resolution.

Kilshtain-Vardi A, Glick M, Greenblatt HM, Goldblum A, Shoham G
Acta Crystallogr D Biol Crystallogr 59 323-33 (2003)
Cited: 18 times
EuropePMC logo PMID: 12554943

Abstract

The crystal structure of the bovine zinc metalloproteinase carboxypeptidase A (CPA) has been refined to 1.25 A resolution based on room-temperature X-ray synchrotron data. The significantly improved structure of CPA at this resolution (anisotropic temperature factors, R factor = 10.4%, R(free) = 14.5%) allowed the modelling of conformational disorders of side chains, improved the description of the protein solvent network (375 water molecules) and provided a more accurate picture of the interactions between the active-site zinc and its ligands. The calculation of standard uncertainties in individual atom positions of the refined model of CPA allowed the deduction of the protonation state of some key residues in the active site and confirmed that Glu72 and Glu270 are negatively charged in the resting state of the enzyme at pH 7.5. These results were further validated by theoretical calculations that showed significant reduction of the pK(a) of these side chains relative to solution values. The distance between the zinc-bound solvent molecule and the metal ion is strongly suggestive of a neutral water molecule and not a hydroxide ion in the resting state of the enzyme. These findings could support both the general acid/general base mechanism, as well as the anhydride mechanism suggested for CPA.

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  4. Structure of Mycobacterium tuberculosis Rv2714, a representative of a duplicated gene family in Actinobacteria. Graña M, Bellinzoni M, Miras I, Fiez-Vandal C, Haouz A, Shepard W, Buschiazzo A, Alzari PM. Acta Crystallogr Sect F Struct Biol Cryst Commun 65 972-977 (2009)
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  2. Activation of zinc-requiring ectoenzymes by ZnT transporters during the secretory process: Biochemical and molecular aspects. Kambe T, Takeda TA, Nishito Y. Arch Biochem Biophys 611 37-42 (2016)

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  1. Zinc transporters, ZnT5 and ZnT7, are required for the activation of alkaline phosphatases, zinc-requiring enzymes that are glycosylphosphatidylinositol-anchored to the cytoplasmic membrane. Suzuki T, Ishihara K, Migaki H, Matsuura W, Kohda A, Okumura K, Nagao M, Yamaguchi-Iwai Y, Kambe T. J Biol Chem 280 637-643 (2005)
  2. Crystal structure of human carboxypeptidase M, a membrane-bound enzyme that regulates peptide hormone activity. Reverter D, Maskos K, Tan F, Skidgel RA, Bode W. J Mol Biol 338 257-269 (2004)
  3. Crystal structure of the LasA virulence factor from Pseudomonas aeruginosa: substrate specificity and mechanism of M23 metallopeptidases. Spencer J, Murphy LM, Conners R, Sessions RB, Gamblin SJ. J Mol Biol 396 908-923 (2010)
  4. Stabilization of internal charges in a protein: water penetration or conformational change? Denisov VP, Schlessman JL, García-Moreno E B, Halle B. Biophys J 87 3982-3994 (2004)
  5. The three-dimensional structures of tick carboxypeptidase inhibitor in complex with A/B carboxypeptidases reveal a novel double-headed binding mode. Arolas JL, Popowicz GM, Lorenzo J, Sommerhoff CP, Huber R, Aviles FX, Holak TA. J Mol Biol 350 489-498 (2005)
  6. NMR studies of zinc binding in a multi-histidinic peptide fragment. Zoroddu MA, Medici S, Peana M, Anedda R. Dalton Trans 39 1282-1294 (2010)
  7. Modelling the structure of latexin-carboxypeptidase A complex based on chemical cross-linking and molecular docking. Mouradov D, Craven A, Forwood JK, Flanagan JU, García-Castellanos R, Gomis-Rüth FX, Hume DA, Martin JL, Kobe B, Huber T. Protein Eng Des Sel 19 9-16 (2006)
  8. Predictive methods for computational metalloenzyme redesign - a test case with carboxypeptidase A. Valdez CE, Morgenstern A, Eberhart ME, Alexandrova AN. Phys Chem Chem Phys 18 31744-31756 (2016)
  9. Crystal structure of bacteriophage ϕNIT1 zinc peptidase PghP that hydrolyzes γ-glutamyl linkage of bacterial poly-γ-glutamate. Fujimoto Z, Kimura K. Proteins 80 722-732 (2012)
  10. Database for three dimensional structures of pepsin-like enzymes using the internal coordinate system. Russ A, Pechik I, Andreeva N. Proteins 61 223-226 (2005)
  11. Spermine as a possible endogenous allosteric activator of carboxypeptidase A: multispectroscopic and molecular simulation studies. Mohammadi M, Shareghi B, Akbar Saboury A, Farhadian S. J Biomol Struct Dyn 38 101-113 (2020)


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