Active site of bee venom phospholipase A2: the role of histidine-34, aspartate-64 and tyrosine-87.
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 pKa 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 pKa 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 pKa is unchanged. These pKas 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.
Annand
R R
RR
Department of Chemistry, University of Washington, Seattle 98195, USA.
Kontoyianni
M
M
Penzotti
J E
JE
Dudler
T
T
Lybrand
T P
TP
Gelb
M H
MH
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001514:Bee Venoms / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D004563:Electrochemistry; D004926:Escherichia coli; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D010741:Phospholipases A / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D054467:Phospholipases A2; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D001514:Bee Venoms; D017931:DNA Primers; D011994:Recombinant Proteins; D010741:Phospholipases A; D054467:Phospholipases A2