Title

Temperature and pH Dependence on Enzymatic Activity of A82F F87A Mutant and Wild-type Cytochrome P450 BM3

Presentation Type

Event

Start Date

27-4-2019 10:50 AM

End Date

27-4-2019 11:29 AM

Abstract

Cytochrome P450 is an enzyme that plays a key role in the breakdown of medicine and toxins. It does this by hydroxylating substrates making the substrates more water soluble. P450s are also involved in cholesterol and hormone synthesis. The third P450 from bacterium Bacillus Megaterium (BM3) is used as a model P450 enzyme due to its structural similarity to human P450s. BM3’s exact function is not known but has been shown to be highly efficient in the hydroxylation of fatty acids. Within the enzyme’s active site, substrates can occupy two conformations, distal or proximal, which can be monitored by UV-vis spectroscopy. The percentage of the substrate in the proximal conformation was found to vary as a function of temperature. A mutant was developed where it was hypothesized that the proximal conformation would be more prevalent. Enzymatic Studies with palmitic (C16:0), lauric (C12:0), and arachidonic (C20:4) acids as substrates were performed for the wild-type and mutant enzyme. Michaelis–Menten Kinetics were used to determine Km, kcat, and catalytic efficiency of the enzymes. The results of the experiments showed the mutant enzyme has higher catalytic efficiency for arachidonic acid compared to the wild-type. However, the increase in catalytic efficiency of the mutant over the wild-type enzyme varies when temperature and pH act as variables. Future studies include looking at the products of the enzymatic reaction as a function of temperature and pH by LC/MS.

This document is currently not available here.

Share

COinS
 
Apr 27th, 10:50 AM Apr 27th, 11:29 AM

Temperature and pH Dependence on Enzymatic Activity of A82F F87A Mutant and Wild-type Cytochrome P450 BM3

Cytochrome P450 is an enzyme that plays a key role in the breakdown of medicine and toxins. It does this by hydroxylating substrates making the substrates more water soluble. P450s are also involved in cholesterol and hormone synthesis. The third P450 from bacterium Bacillus Megaterium (BM3) is used as a model P450 enzyme due to its structural similarity to human P450s. BM3’s exact function is not known but has been shown to be highly efficient in the hydroxylation of fatty acids. Within the enzyme’s active site, substrates can occupy two conformations, distal or proximal, which can be monitored by UV-vis spectroscopy. The percentage of the substrate in the proximal conformation was found to vary as a function of temperature. A mutant was developed where it was hypothesized that the proximal conformation would be more prevalent. Enzymatic Studies with palmitic (C16:0), lauric (C12:0), and arachidonic (C20:4) acids as substrates were performed for the wild-type and mutant enzyme. Michaelis–Menten Kinetics were used to determine Km, kcat, and catalytic efficiency of the enzymes. The results of the experiments showed the mutant enzyme has higher catalytic efficiency for arachidonic acid compared to the wild-type. However, the increase in catalytic efficiency of the mutant over the wild-type enzyme varies when temperature and pH act as variables. Future studies include looking at the products of the enzymatic reaction as a function of temperature and pH by LC/MS.