title: Hemiacetal stabilization in a chymotrypsin inhibitor complex and the reactivity of the hydroxyl group of the catalytic serine residue of chymotrypsin.
abstract: The aldehyde inhibitor Z-Ala-Ala-Phe-CHO has been synthesized and shown by (13)C-NMR to react with the active site serine hydroxyl group of alpha-chymotrypsin to form two diastereomeric hemiacetals. For both hemiacetals oxyanion formation occurs with a pKa value of ~7 showing that chymotrypsin reduces the oxyanion pKa values by ~5.6 pKa units and stabilizes the oxyanions of both diastereoisomers by ~32kJmol(-1). As pH has only a small effect on binding we conclude that oxyanion formation does not have a significant effect on binding the aldehyde inhibitor. By comparing the binding of Z-Ala-Ala-Phe-CHO with that of Z-Ala-Ala-Phe-H we estimate that the aldehyde group increases binding ~100 fold. At pH7.2 the effective molarity of the active site serine hydroxy group is ~6000 which is ~7× less effective than with the corresponding glyoxal inhibitor. Using (1)H-NMR we have shown that at both 4 and 25°C the histidine pKa is ~7.3 in free chymotrypsin and it is raised to ~8 when Z-Ala-Ala-Phe-CHO is bound. We conclude that oxyanion formation only has a minor role in raising the histidine pKa and that the aldehyde hydrogen must be replaced by a larger group to raise the histidine pKa>10 and give stereospecific formation of tetrahedral intermediates. The results show that a large increase in the pKa of the active site histidine is not needed for the active site serine hydroxyl group to have an effective molarity of 6000.
authors.affiliation: School of Biomolecular and Biomedical Science, Centre for Synthesis and Chemical Biology, Conway Institute, University College Dublin, Dublin 4, Ireland.
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Cleary
authors.affiliation: School of Chemistry and Chemical Biology, Centre for Synthesis and Chemical Biology, Conway Institute, University College Dublin, Dublin 4, Ireland.
authors.forename: William
authors.initials: W
authors.lastname: Doherty
authors.affiliation: School of Chemistry and Chemical Biology, Centre for Synthesis and Chemical Biology, Conway Institute, University College Dublin, Dublin 4, Ireland.
authors.forename: Paul
authors.initials: P
authors.lastname: Evans
authors.affiliation: School of Biomolecular and Biomedical Science, Centre for Synthesis and Chemical Biology, Conway Institute, University College Dublin, Dublin 4, Ireland. Electronic address: J.Paul.G.Malthouse@ucd.ie.
authors.forename: J Paul G
authors.initials: JP
authors.lastname: Malthouse
mesh_terms: D000818:Animals; D055162:Biocatalysis; D020134:Catalytic Domain; D002417:Cattle; D002918:Chymotrypsin / Q000737:chemistry*; D006037:Glyoxal / Q000737:chemistry; D006639:Histidine / Q000737:chemistry*; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D019906:Nuclear Magnetic Resonance, Biomolecular; D009842:Oligopeptides / Q000138:chemical synthesis / Q000737:chemistry*; D011480:Protease Inhibitors / Q000138:chemical synthesis / Q000737:chemistry*; D012694:Serine / Q000737:chemistry*; D012996:Solutions; D013237:Stereoisomerism; D013816:Thermodynamics
chemical_list: D009842:Oligopeptides; D011480:Protease Inhibitors; D012996:Solutions; D012694:Serine; D006639:Histidine; D006037:Glyoxal; D002918:Chymotrypsin; C117608:alpha-chymotrypsin
keywords: Aldehyde inhibitor; Chymotrypsin; Effective molarity; Hemiacetal; Oxyanion