Catalytic cycle of the phosphatidylcholine-preferring phospholipase C from Bacillus cereus. Solvent viscosity, deuterium isotope effects, and proton inventory studies.
The phosphatidylcholine-preferring phospholipase C from Bacillus cereus (PLCBc) is a 28.5 kDa enzyme with three zinc ions in its active site. Although much is known about the roles that various PLCBc active site amino acids play in binding and catalysis, there is little information about the rate-determining step of the PLCBc-catalyzed hydrolysis of phospholipids and the catalytic cycle of the enzyme. To gain insight into these aspects of the hydrolysis, solvent viscosity variation experiments were conducted to determine whether an external step (substrate binding or product release) or an internal step (hydrolysis) is rate-limiting. The data indicate that the PLCBc-catalyzed reaction is unaffected by changes in solvent viscosity. This observation is inconsistent with the notion of substrate binding or product release being rate-determining and supports the hypothesis that a chemical step is rate-limiting. Furthermore, a deuterium isotope effect of 1.9 and a linear proton inventory plot indicate one proton is transferred in the rate-determining step. These data may be used to formulate a comprehensive catalytic cycle that is for the first time based on experimental evidence. In this mechanism, Asp55 of PLCBc activates an active site water molecule for attack on the phosphodiester bond, the hydrolysis of which is rate-limiting. The phosphorylcholine product is the first to leave the active site, followed by diacylglycerol.
Martin
S F
SF
Department of Chemistry and Biochemistry, University of Texas at Austin 78712, USA. sfmartin@mail.utexas.edu
Hergenrother
P J
PJ
D001409:Bacillus cereus / Q000201:enzymology*; D001665:Binding Sites; D002384:Catalysis; D003903:Deuterium / Q000737:chemistry; D006868:Hydrolysis; D010713:Phosphatidylcholines / Q000737:chemistry*; D011522:Protons; D012997:Solvents; D013379:Substrate Specificity; D010738:Type C Phospholipases / Q000737:chemistry*; D014783:Viscosity
D010713:Phosphatidylcholines; D011522:Protons; D012997:Solvents; D003903:Deuterium; D010738:Type C Phospholipases