BIOCHEM 1: a. Using Excel or similar software, make two diagnostic Lineweaver-Bu

Question

BIOCHEM 1:

a. Using Excel or similar software, make two diagnostic Lineweaver-Burk plots: one for methanol and another for the other three alcohols. Include coordinate values in a table and show the trendline equations on the plots.

b. What are the systematic name and E.C. classification number for this enzyme?

c. Is ADH a Michaelis-Menton enzyme? What is the basis for your answer?

d. What are the Km and kcat values for each of the substrates?

e. If the k1 and k-1 bisubstrate reaction rate constants ethanol (NB these can be treated as monosubstrate reaction rate constants k1 and k-1 with saturating concentrations of NAD+ ) are 12.4 x105 M-1 s -1 and 560 s -1 , respectively, is ADH a rapid equilibrium enzyme or does it only follow steady state kinetics? What is the basis for your answer?

f. Calculate the catalytic efficiencies with each of the alcohols and rank them in order of decreasing efficiency.

g. What can you conclude determines efficiency of ADH for ethanol, n-propanol and n-butanol? State your rationale. What does your conclusion say about the structure of the active site of the enzyme?

h. What do the kcat values for ethanol, n-propanol and n-butanol say about the structure of the active site?

i. What can you conclude about the binding of methanol in the active site?

j. Is ADH a perfect enzyme with the best of these substrates? What is the basis for your answer?

Human class 1 liver alcohol dehydrogenase (ADH) is a homodimer with two catalytic sites per enzyme. It catalyzes the following reversible reaction R-OH + NAD+ R'=O + NADH + H The catalysis follows an ordered single displacement mechanism with NAD binding first. During a kinetic analysis of this ADH the following data were obtained using different alcohols and a saturating concentration of NAD as substrates R-OH oxidation by human class 1 ADH uM R-OH n-butanol methanol 0.0114 0.0284 0.056 ethanol 1.22 2.93 5.5 9.78 18.3 n-propanol 20 50 100 200 500 2.85 6.38 10.9 16.8 25 13.3 22.6 29.4 34.6 38.7 0.267

Explanation / Answer

b. The systematic name of the enzyme is alcohol:NAD+ oxidoreductase and the E.C classification number is EC 1.1.1.1

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