Interfacial catalysis by phospholipase A sub 2 : Evaluation of the interfacial rate constants by steady-state isotope effect studies

Journal Article · Tue Jul 23 04:00:00 EDT 1991 · Biochemistry; (United States)

DOI:https://doi.org/10.1021/bi00243a035· OSTI ID:5080762

Ghomashchi, F; O'Hare, T; Clary, D; Gelb, M H ^[1]

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1. Univ. of Washington, Seattle (United States)

The kinetics of hydrolysis of phospholipid vesicles by phospholipase A{sub 2}, (PLA2) in the scooting mode can be described by the Michaelis-Menten formalism for the action of the enzyme in the interface (E*). E* + S {l equilibrium} E*S {l equilibrium} E*P {l equilibrium} E* + Products The values of the interfacial rate constants cannot be obtained by classical methods because the concentration of the substrate within the lipid bilayer is not easily manipulated. In the repesent study, carbonyl-carbon heavy atom isotope effects for the hydrolysis of phospholipids have been measured in both vesicles and in mixed micelles in which the phospholipid was present in the nonionic detergent Triton X-100. A large ({sup 14}C)carbonyl carbon isotope effect of 1.12{plus minus}0.02 was measured for the cobra venom PLA2-catalyzed hydrolysis of dipalmitoylphosphatidylcholine in Triton X-100. In contrast, no isotope effect was measured for the action of the porcine pancreatic and cobra venom enzymes on vesicles of dimyrisstoylphosphatidylmethanol in the scooting mode. In a second experiment, the hydrolysis of vesicles was carried out in oxygen-18 enriched water. Analysis of the released fatty acid product by mass spectrometry showed that it contained only a single oxygen-18. All of these results were used to estimate both the forward and reverse commitments to catalysis.

• OSTI ID: 5080762
• Journal Information: Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 30:29; ISSN 0006-2960; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
BIOCHEMICAL REACTION KINETICS
CARBON 14 COMPOUNDS
CARBON COMPOUNDS
CARBOXYLESTERASES
CHEMICAL REACTIONS
DECOMPOSITION
ENZYMATIC HYDROLYSIS
ENZYMES
ESTERASES
ESTERS
EVEN-EVEN NUCLEI
HYDROLASES
HYDROLYSIS
ISOTOPE EFFECTS
ISOTOPES
KINETICS
LABELLED COMPOUNDS
LIGHT NUCLEI
LIPASES
LIPIDS
LYSIS
NUCLEI
ORGANIC COMPOUNDS
ORGANIC PHOSPHORUS COMPOUNDS
OXYGEN 18
OXYGEN ISOTOPES
PHOSPHOLIPIDS
REACTION KINETICS
SOLVOLYSIS
STABLE ISOTOPES