Kinetic analysis of yeast phosphatidate phosphatase toward Triton X-100/phosphatidate mixed micelles.
A detailed kinetic analysis of purified yeast membrane-associated phosphatidate phosphatase was performed using Triton X-100/phosphatidate mixed micelles. Enzyme activity was dependent on the bulk and surface concentrations of phosphatidate. These results were consistent with the "surface dilution" kinetic scheme (Deems, R. A., Eaton, B. R., and Dennis, E. A. (1975) J. Biol. Chem. 250, 9013-9020) where phosphatidate phosphatase binds to the mixed micelle surface before binding to its substrate and catalysis occurs. Phosphatidate phosphatase was shown to physically associate with Triton X-100 micelles in the absence of phosphatidate, however, the enzyme was more tightly associated with micelles when its substrate was present. The enzyme had 5- to 6-fold greater affinity (reflected in the dissociation constant nKsA/chi) for Triton X-100 micelles containing dioleoyl-phosphatidate and dipalmitoyl-phosphatidate when compared to micelles containing dicaproyl-phosphatidate. The Vmax for dioleoyl-phosphatidate was 3.8-fold higher than the Vmax for dipalmitoyl-phosphatidate, whereas the interfacial Michaelis constant chi KmB for dipalmitoyl-phosphatidate was 3-fold lower than the chi KmB for dioleoyl-phosphatidate. The specificity constants (Vmax/chi KmB) of both substrates were similar which indicated that dioleoyl-phosphatidate and dipalmitoyl-phosphatidate were equally good substrates. Based on catalytic constants (Vmax and chi KmB), dicaproyl-phosphatidate was the best substrate with an 11- and 14-fold greater specificity constant when compared to dioleoyl-phosphatidate and dipalmitoyl-phosphatidate, respectively.
Lin
Y P
YP
Department of Food Science, Cook College, New Jersey Agricultural Experiment Station, Rutgers University, New Brunswick 08903.
Carman
G M
GM
D002384:Catalysis; D002845:Chromatography; D003102:Colloids; D007700:Kinetics; D008823:Micelles; D017830:Octoxynol; D010711:Phosphatidate Phosphatase / Q000378:metabolism*; D010712:Phosphatidic Acids / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D011092:Polyethylene Glycols; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013379:Substrate Specificity
D003102:Colloids; D008823:Micelles; D010712:Phosphatidic Acids; C037657:dioleoylphosphatidic acid; C031599:dipalmitoylphosphatidic acid; D011092:Polyethylene Glycols; D017830:Octoxynol; C062215:dicaproylphosphatidic acid; D010744:Phosphoric Monoester Hydrolases; D010711:Phosphatidate Phosphatase