Using X-ray crystallography of the Asp55Asn mutant of the phosphatidylcholine-preferring phospholipase C from Bacillus cereus to support the mechanistic role of Asp55 as the general base.
Because mutations of the ionizable Asp at position 55 of the phosphatidylcholine preferring phospholipase C from Bacillus cereus (PLC(Bc)) to a non-ionizable Asn generate a mutant enzyme (D55N) with 10(4)-fold lower catalytic activity than the wild-type enzyme, we tentatively identified Asp55 as the general base for the enzymatic reaction. To eliminate the alternate possibility that Asp55 is a structurally important amino acid, the X-ray structures of unbound D55N and complexes of D55N with two non-hydrolyzable substrate analogues have been solved and refined to 2.0, 2.0, and 2.3A, respectively. The structures of unbound wild-type PLC(Bc) and a wild-type PLC(Bc)-complex with a non-hydrolyzable substrate analogue do not change significantly as a result of replacing Asp55 with Asn. These observations demonstrate that Asp55 is not critical for the structural integrity of the enzyme and support the hypothesis that Asp55 is the general base in the PLC(Bc)-catalyzed hydrolysis of phospholipids.
Antikainen
Nina M
NM
Department of Chemistry and Biochemistry, The Institute of Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX 78712, USA.
Monzingo
Arthur F
AF
Franklin
Christopher L
CL
Robertus
Jon D
JD
Martin
Stephen F
SF
D019943:Amino Acid Substitution; D001224:Aspartic Acid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001409:Bacillus cereus / Q000201:enzymology*; D002384:Catalysis; D018360:Crystallography, X-Ray; D018698:Glutamic Acid / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D016297:Mutagenesis, Site-Directed; D010713:Phosphatidylcholines / Q000378:metabolism*; D017354:Point Mutation; D011487:Protein Conformation; D013379:Substrate Specificity; D010738:Type C Phospholipases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015032:Zinc / Q000737:chemistry / Q000378:metabolism
D008024:Ligands; D010713:Phosphatidylcholines; D001224:Aspartic Acid; D018698:Glutamic Acid; D010738:Type C Phospholipases; D015032:Zinc