Structural Consequences of Hydrophilic Amino Acid Substitutions in the Hydrophobic Pocket of Human Carbonic Anhydrase II

Satish K. Nair, David W. Christianson

Research output: Contribution to journalArticle

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 Å. 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. The so-called “deep” water molecule, which resides at the mouth of the hydrophobic pocket in a location believed to be the precatalytic association site for substrate, is not displaced in any of the variants. Since the structure of the CO2 association site is not severely compromised, it is reasonable that catalytic efficiency is not severely compromised in Leu-198 variants. Finally, the effects of residue 198 on structure-activity relationships of nucleophilic zinc-bound hydroxide are minimal, except in the Leu-198→Glu variant.

Original languageEnglish (US)
Pages (from-to)4506-4514
Number of pages9
JournalBiochemistry
Volume32
Issue number17
DOIs
StatePublished - May 1 1993

ASJC Scopus subject areas

  • Biochemistry

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