KINETICS OF OXIDATION OF TYROSINE AND DITYROSINE BY MYELOPEROXIDASE COMPOUND-I AND COMPOUNDS-II - IMPLICATIONS FOR LIPOPROTEIN PEROXIDATIONSTUDIES

The oxidation of lipoproteins is considered to play a key role in athe rogenesis, and tyrosyl radicals have been implicated in the oxidation reaction. Tyrosyl radicals are generated in a system containing myelop eroxidase, H2O2, and tyrosine, but details of this enzyme-catalyzed re action have not been explored, We have performed transient spectral an d kinetic measurements to study the oxidation of tyrosine by the myelo peroxidase intermediates, compounds I and II, using both sequential mi xing and single-mixing stopped-flow techniques. The one electron reduc tion of compound I to compound II by tyrosine has a second order rate constant of (7.7 +/- 0.1) x 10(5) M(-1) s(-1). Compound II is then red uced by tyrosine to native enzyme with a second order rate constant of (1.57 +/- 0.06) x 10(4) M(-1) s(-1). Our study further revealed that, compared with horseradish peroxidase, thyroid peroxidase, and lactope roxidase, myeloperoxidase is the most efficient catalyst of tyrosine o xidation at physiological pH. The second order rate constant for the m yeloperoxidase compound I reaction with tyrosine is comparable with th at of its compound I reaction with chloride: (4.7 +/- 0.1) x 10(6) M(- 1) s(-1). Thus, although chloride is considered the major myeloperoxid ase substrate, tyrosine is able to compete effectively for compound I. Steady state inhibition studies demonstrate that chloride binds very weakly to the tyrosine binding site of the enzyme. Coupling of tyrosyl radicals yields dityrosine, a highly fluorescent stable compound that had been identified as a possible marker for lipoprotein oxidation. W e present spectral and kinetic data showing that dityrosine is further oxidized by both myeloperoxidase compounds I and II. The second order rate constants we determined for dityrosine oxidation are (1.12 +/- 0 .01) x 10(5) M(-1) s(-1) for compound I and (7.5 +/- 0.3) x 10(2) M(-1 ) s(-1) for compound II. Therefore, caution must be exercised when usi ng dityrosine as a quantitative index of lipoprotein oxidation, partic ularly in the presence of myeloperoxidase and H2O2.