(1) (a) Describe how you would obtain kinetic parameters (K a ,K b ,V max ,K ia ,K ib ) in a sequential two substrate enzyme-catalysed reaction.

(b) Penicillin is hydrolysed and thereby rendered inactive by penicillinase (also known as 5- lactamase), an enzyme present in some resistant bacteria. The mass of this enzyme in Staphylococcus aureus is 29.6 kDal. The amount of penicillin hydrolysed in 1 minute in a 10-ml solution containing 10 ^ - 90 g of purified penicillinase was measured as function of the concentration of penicillin. Assume that the concentration does not change appreciably during the assay.

[Penicillin] (µM)

Amount hydrolysed (nanomoles)

1

0.11

3

0.25

5

0.34

10

0.45

30

0.58

50

0.61

(i) Plot V_{0} versus [S] and 1 / V_{c} versus 1 / [S] for these data. Does penicillinase appear to obey Michaelis-Menten kinetics? If so what is the value of K_{m}

(ii) What is the value of V max ^ *

(iii) What is the turnover number of penicillinase under these experimental conditions? Assume one active site per enzyme molecule.

(iv)How long does it take the enzyme for one round of catalysis?