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Lecture 1 - enzyme kinetics

Estell et al 1985 engineering an enzyme by site

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Unformatted text preview: 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-Ala100 Enzyme Kinetics! Enzyme Assays - effects of time! Enzyme Assays and Measurements of Reaction Rates Replicate test tubes (Rxns.) 1.  Mix Substrate with buffer solution (1) Mix S together with pH buffer solution in a test tube Replicate tubes contain same total volume 2.  Add Enzyme to start reaction S ALWAYS present in VAST EXCESS over E 3.  Incubate @ specific T (and pH) EXCESS ° SATURATING ( .  optimal for enzyme, unless r 4pH Measure in P oeffect ofipHS being studied) n is with time (2) Add E to start reaction (time = 0) 5.  Plot progress curve V0 (slope of line near time =0) (3) Incubate at a temperature that is optimal for the E (4) Measure increase in P or decrease in S with time, calculate reaction rate Monitor progress of reaction. Possible...
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