IN-VITRO EVOLUTION OF HORSE HEART MYOGLOBIN TO INCREASE PEROXIDASE-ACTIVITY

Random mutagenesis and screening for enzymatic activity has been used to engineer horse heart myoglobin to enhance its intrinsic peroxidase activity. A chemically synthesized gene encoding horse heart myoglobin was subjected to successive cycles of PCR random mutagenesis, The mut ated myoglobin gene was expressed in Escherichia coli LE392, and the v ariants were screened for peroxidase activity with a plate assay. Four cycles of mutagenesis and screening produced a series of single, doub le, triple, and quadruple variants with enhanced peroxidase activity. Steady-state kinetics analysis demonstrated that the quadruple variant T39I/K45D/F46L/I107F exhibits peroxidase activity significantly great er than that of the wild-type protein with k(1) (for H2O2 oxidation of metmyoglobin) of 1.34 x 10(4) M-1 s(-1) (approximate to 25-fold that of wild-type myoglobin) and ks [for reducing the substrate (2, 2'-azin o-di-(3-ethyl)benzthiazoline-6-sulfonic acid] of 1.4 x 10(6) M-1 s(-1) (1.6-fold that of wild-type myoglobin), Thermal stability of these va riants as measured with circular dichroism spectroscopy demonstrated t hat the T-m of the quadruple variant is decreased only slightly compar ed with wild-type (74.1 degrees C vs. 76.5 degrees C), The rate consta nts for binding of dioxygen exhibited by the quadruple variant are ide ntical to the those observed for wild-type myoglobin (k(on), 22.2 x 10 (-6) M-1 s(-1) vs. 22.3 x 10(-6) M-1 s(-1); k(off), 24.3 s(-1) vs. 24. 2 s(-1); K-O2, 0.91 x 10(-6) M-1 vs. 0.92 x 10(-6) M-1). The affinity of the quadruple variant for CO is increased slightly (k(on), 0.90 x 1 0(-6) M-1 s(-1) vs. 0.51 x 10(-6) M-1 s(-1); k(off), 5.08 s(-1) vs. 3. 51 s(-1); K-CO, 1.77 x 10(-7) M-1 vs. 1.45 x 10(-7) M-1). All four sub stitutions are in the heme pocket and within 5 Angstrom of the heme gr oup.