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Unformatted text preview: 1985. Engineering an Enzyme by Site-directed Mutagenesis to Be Resistant to Chemical Oxidation.
T o determine t he s. Biol. Chem. 260: 6518-6521.
Jpecific activity of each mutant enzyme,
enzymes were purified from culture supernatants, and their
concentrations were determined spectrophotometrically. T he
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
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.
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- Summer '09