Active Site of Bee Venom Phospholipase A2:  The Role of Histidine-34, Aspartate-64 and Tyrosine-87

• Published in: Biochemistry (Easton) Vol. 35; no. 14; pp. 4591 - 4601
• Main Authors: Annand, Robert R, Kontoyianni, Maria, Penzotti, Julie E, Dudler, Thomas, Lybrand, Terry P, Gelb, Michael H
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 09-04-1996
• Subjects: Amino Acid Sequence; Animals; Base Sequence; Bee Venoms - chemistry; Bee Venoms - enzymology; Bee Venoms - genetics; Binding Sites - genetics; DNA Primers - genetics; Electrochemistry; Escherichia coli; Hydrogen Bonding; Hydrogen-Ion Concentration; In Vitro Techniques; Kinetics; Molecular Sequence Data; Molecular Structure; Mutagenesis, Site-Directed; Phospholipases A - chemistry; Phospholipases A - genetics; Phospholipases A - metabolism; Phospholipases A2; Protein Conformation; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi9528412

In bee venom phospholipase A2, histidine-34 probably functions as a Brønsted base to deprotonate the attacking water. Aspartate-64 and tyrosine-87 form a hydrogen bonding network with histidine-34. We have prepared mutants at these positions and studied their kinetic properties. The mutant in which histidine-34 is changed to glutamine is catalytically inactive, while the mutants in which aspartate-64 is changed to asparagine or alanine (interfacial turnover numbers are reduced by 50−100-fold) or in which tyrosine-87 is changed to phenylalanine (no change in turnover number) retain good activity. The interfacial Michaelis constants are changed by less than 10-fold for all mutants. Molecular simulations suggest that mutation of aspartate-64 and tyrosine-87 should yield enzymes that retain a native-like structure and support catalysis. The pK a of the histidine-34 imidazole was deduced from the pH-rate profile and from the pH dependence of the rate of histidine-34 alkylation by 2-bromo-4‘-nitroacetophenone. The pK a is increased about one-half unit by the tyrosine-87 mutation and reduced about one-half unit by the aspartate-64 to asparagine mutation, while in the aspartate-64 to alanine mutant the pK a is unchanged. These pK as are generally consistent with results of electrostatic calculations and suggest that the hydrogen bond between aspartate-64 and histidine-34 is not unusually strong. The hydrogen bonding network linking tyrosine-87 to aspartate-64 and aspartate-64 to histidine-34 is not critical for catalysis.