Lecture 1 - enzyme kinetics

Furthermore possible recombination between t he

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Unformatted text preview: een t he plasmid containing the mutant subtilisin gene and the host subtilisin gene can be avoided. Estell et al., 1985. Engineering an Enzyme by Site-directed Mutagenesis to Be Resistant to Chemical Oxidation. Relative specific T o determine t he s. Biol. Chem. 260: 6518-6521. Jpecific activity of each mutant enzyme, activity enzymes were purified from culture supernatants, and their % concentrations were determined spectrophotometrically. T he 138 enzymes were assayed against t he substrate, succinyl-L-Ala- ities of codon 222 m utant subtilisins purified a nd assayed a s described under 100 Enzyme Kinetics! Case Study - Genetic Engineering of Subtilisin! Table II: Kinetic Constants for selected Codon 222 mutants Codon 222 Kcat (S-1) Km (M) Kcat / Km (M-1 S-1) Met (wild-type) Cys Ser Leu 50 84 27 5 1.4 x 10-4 4.8 x 10-4 6.3 x 10-4 2.6 x 10-4 36x104 20x104 4x104 2x104 Enzyme Kinetics! Getting numbers from Reaction Curves! Although Vmax can t be measured directly, its value can be obtained by extrapolation. The vo versus [S] curve is a rectangular hyperbola...
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