The importance of glycine-30 for enzymatic activity of phospholipase A2.
The nearly conserved glycine-30 in porcine pancreatic phospholipase A2 has been replaced by serine. The resulting mutant G30S was expressed in Escherichia coli, purified and characterized. The mutation caused a significant drop in enzymatic activity towards monomeric and aggregated substrates, but had a limited effect on substrate binding. In contrast the affinity for calcium ions, the essential cofactor, was reduced 10-fold. The reduced enzymatic activity is attributed to a reduced stabilization of the transition state. The results are discussed in view of naturally occurring inactive phospholipase A2 homologues from snake venom.
Bekkers
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AC
Department of Enzymology and Protein Engineering, University of Utrecht, CBLE, University Centre De Uithof, The Netherlands.
Franken
P A
PA
Toxopeus
E
E
Verheij
H M
HM
de Haas
G H
GH
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D005998:Glycine; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015345:Oligonucleotide Probes; D010179:Pancreas / Q000201:enzymology; D010741:Phospholipases A / Q000235:genetics / Q000378:metabolism*; D054467:Phospholipases A2; D015183:Restriction Mapping; D013379:Substrate Specificity; D013552:Swine
D015345:Oligonucleotide Probes; D010741:Phospholipases A; D054467:Phospholipases A2; D005998:Glycine