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Structural consequences of hydrophilic amino acid substitutions in the hydrophobic pocket of human carbonic anhydrase II.

Nair SK, Christianson DW
Biochemistry 32 4506-14 (1993)
Related entries: 1hea, 1heb, 1hed

Cited: 7 times
EuropePMC logo PMID: 8485129

Abstract

The three-dimensional structures of Leu-198-->Glu, Leu-198-->His, Leu-198-->Arg, and Leu-198-->Ala variants of human carbonic anhydrase II (CAII) have each been determined by X-ray crystallographic methods to a resolution of 2.0 A. The side chain of Leu-198 is located at the mouth of the active site hydrophobic pocket, and this pocket is required for substrate association. Hydrophobic-->hydrophilic amino acid substitutions at the mouth of the pocket decrease kcat/KM for CO2 hydration: the CO2 hydrase activities of Leu-198-->Glu, Leu-198-->His, and Leu-198-->Arg CAIIs are diminished 19-fold, 10-fold, and 17-fold, respectively, relative to the wild-type enzyme; however, the substitution of a compact aliphatic side chain for Leu-198 has a smaller effect on catalysis, in that Leu-198-->Ala CAII exhibits only a 3-fold decrease in CO2 hydrase activity [Krebs, J. F., Rana, F., Dluhy, R. A., & Fierke, C. A. (1993) Biochemistry (preceding paper in this issue)]. It is intriguing that CO2 hydrase activity is not severely diminished in Leu-198-->Arg CAII, even though the side chain of Arg-198 blocks the hydrophobic pocket. Therefore, the bulky side chain of Arg-198 must be reasonably mobile in order to accommodate substrate association. Significantly, a residue larger than the wild-type Leu-198 side chain does not necessarily block the substrate association pocket; e.g., the side chain of Glu-198 packs against a hydrophobic patch, the net result of which is a wider mouth for the pocket.(ABSTRACT TRUNCATED AT 250 WORDS)

Reviews citing this publication (3)

  1. Carbonic anhydrase as a model for biophysical and physical-organic studies of proteins and protein-ligand binding. Krishnamurthy VM, Kaufman GK, Urbach AR, Gitlin I, Gudiksen KL, Weibel DB, Whitesides GM. Chem Rev 108 946-1051 (2008)
  2. Catalysis by metal-activated hydroxide in zinc and manganese metalloenzymes. Christianson DW, Cox JD. Annu Rev Biochem 68 33-57 (1999)
  3. Thermodynamic, kinetic, and structural parameterization of human carbonic anhydrase interactions toward enhanced inhibitor design. Linkuvienė V, Zubrienė A, Manakova E, Petrauskas V, Baranauskienė L, Zakšauskas A, Smirnov A, Gražulis S, Ladbury JE, Matulis D. Q Rev Biophys 51 e10 (2018)

Articles citing this publication (4)

  1. Structural study of X-ray induced activation of carbonic anhydrase. Sjöblom B, Polentarutti M, Djinovic-Carugo K. Proc Natl Acad Sci U S A 106 10609-10613 (2009)
  2. Kinetic and structural characterization of thermostabilized mutants of human carbonic anhydrase II. Fisher Z, Boone CD, Biswas SM, Venkatakrishnan B, Aggarwal M, Tu C, Agbandje-McKenna M, Silverman D, McKenna R. Protein Eng Des Sel 25 347-355 (2012)
  3. Replacements in a conserved leucine cluster in the hydrophobic heme pocket of cytochrome c. Lo TP, Murphy ME, Guillemette JG, Smith M, Brayer GD. Protein Sci 4 198-208 (1995)
  4. Structural insights into the effect of active-site mutation on the catalytic mechanism of carbonic anhydrase. Kim JK, Lee C, Lim SW, Andring JT, Adhikari A, McKenna R, Kim CU. IUCrJ 7 985-994 (2020)


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