Question

Determine the kinetic parameters, Km & Vmax and calculate k2.

Penicillin is hydrolyzed and thereby rendered inactive by penicillinase, an enzyme present in some penicillin-resistant bacteria. The mass of this enzyme is 29.5 kD. The amount of penicillin hydrolyzed in 1 minute in a 10 mL solution containing 100 g of purified penicillinase was measured as a function of the concentration of penicillin. Assume that the concentration of penicillin does not change appreciably during the assay. (a) Plot v versus. (Penicillin) and 1/v versus 1/[Penicillin), (b) Determine the kinetic parameters, Km & Vmax. and (c) calculate kz. Substrate = Pennicillin [Penicillin] x-axis 1/[Pennicillin y-axis 1/v UM 1.0 Velocity (W) = Amount hydrolyzed (nM/min) 0.11 0.25 0.34 0.45 0.58 3.0 5.0 10.0 30.0 0.61 50.0

Answer 1

Binal Iniencept = 24 x 0.12 2.4

Slope AV intercept 291 0.12 2.9

D a ll........ . . DATA- /Penicillin lum-) / ( min Inm) 9.09 50-33 0.2 2.2 1.7 0.033 1.6 0.02 CALCULATIONS V = [s] l-michaelis Mertens equation) y intercept slope min nm = lb. 6 mins um " Slope = 16.6 3 mm = 16.6 mins x 10tm 10-17 = 16.6 X 10 mins - km = 16.6x10² mina. Y Intercept & 2.9 mins nm- al = 2.9 mins nm-1. max a imas 10:34 X10-9 M mins- km = 16.6 X 10² mins x max = 18.6 x 103 mins x 0.34 x 10-9 m mir 5.6 X10-6 M. . ANSWER - Km = 5.6 x 10 bm 2 5.6 MM 34 109m = 0.34 minst M may = 0.34

L Concentration of enzyme a no. of moles of enzyme hydrolysed. xos) e 3.3 X 10-14 0:34 X10-4 m et --4-3*16=14 - 210064 mine Turnover number k = laus)

1/1 (1/v) vs (1/penicillin) Linear ((1/v) vs (1/penicillin)) 0.5 1/(penicillin)
This is the plot in Excel.