No abstract

The observed pseudo-first order rate constant for the reaction between a horseradish peroxidase (HRP) variant (R38L)HRPC* and hydrogen peroxide saturates at high peroxide concentrations (Km = 11. 8 mm). The data are consistent with a two-step mechanism involving the formation of an HRP-H2O2 intermediate (k = 1.1 x 10(4) m-1 s-1) whose conversion to compound I is rate-limiting (k = 142 s-1) suggesting that Arg-38 is not only involved in the cleavage of the O-O bond of peroxide but also has an important role in facilitating the rapid binding of H2O2 to HRP. Rapid-scan spectrophotometry revealed the presence of a transient intermediate with a spectrum consistent with a ferric-hydroperoxy complex. At high peroxide concentrations (>500 microM), compound I is converted to compound III without the accumulation of compound II. Spectrophotometric titrations show that arginine 38 is also involved in modulating the apparent affinity of HRPC for reducing substrates such as guaiacol and p-cresol. The spectrum of the complex formed when these substrates bind to the ferric form of the mutant enzyme differs from that observed when they bind to the wild-type ferric enzyme. At neutral and alkaline pH compound I of (R38L)HRPC* was stable and reduced to ferric enzyme without apparent formation of compound II upon titration with p-cresol or ascorbic acid, suggesting a change in the rate-limiting step in the peroxidase cycle. Steady-state kinetic analyses carried out at pH 7.0 showed significant increases in the apparent Km for guaiacol, p-cresol, and 2, 2'-azinobis(3-ethylbenzothiazolinesulfonic acid) (ABTS). The high stability of the oxyferryl form of (R38L)HRPC* and its low catalytic constant for reducing substrates also shows that arginine 38 modulates the reactivity of HRP compound I.

Transient kinetic analysis of biphasic, single turnover data for the reaction of 2,2'-azino-bis[3-ethylbenzthiazoline-6-sulfonic acid] (ABTS) with horseradish peroxidase (HRPC) compound II demonstrated preequilibrium binding of ABTS (k(+5) = 7.82 x 10(4) M(-)(1) s(-)(1)) prior to rate-limiting electron transfer (k(+6) = 42.1 s(-)(1)). These data were obtained using a stopped-flow method, which included ascorbate in the reaction medium to maintain a low steady-state concentration of ABTS (pseudo-first-order conditions) and to minimize absorbance changes in the Soret region due to the accumulation of ABTS cation radicals. A steady-state kinetic analysis of the reaction confirmed that the reduction of HRPC compound II by this substrate is rate-limiting in the complete peroxidase cycle. The reaction of HRPC with o-diphenols has been investigated using a chronometric method that also included ascorbate in the assay medium to minimize the effects of nonenzymic reactions involving phenol-derived radical products. This enabled the initial rates of o-diphenol oxidation at different hydrogen peroxide and o-diphenol concentrations to be determined from the lag period induced by the presence of ascorbate. The kinetic analysis resolved the reaction of HRPC compound II with o-diphenols into two steps, initial formation of an enzyme-substrate complex followed by electron transfer from the substrate to the heme. With o-diphenols that are rapidly oxidized, the heterolytic cleavage of the O-O bond of the heme-bound hydrogen peroxide (k(+2) = 2.17 x 10(3) s(-)(1)) is rate-limiting. The size and hydrophobicity of the o-diphenol substrates are correlated with their rate of binding to HRPC, while the electron density at the C-4 hydroxyl group predominantly influences the rate of electron transfer to the heme.

A naturally occurring gallated polyphenol isolated from green tea leaves, (À À À)-epigallocatechin gallate (EGCG), has been shown to be an inhibitor of dihydrofolate reductase (DHFR) activity in vitro at concentrations found in the serum and tissues of green tea drinkers (0.1-1.0 Mmol/L). These data provide the first evidence that the prophylactic effect of green tea drinking on certain forms of cancer, suggested by epidemiologic studies, is due to the inhibition of DHFR by EGCG and could also explain why tea extracts have been traditionally used in ''alternative medicine'' as anticarcinogenic/antibiotic agents or in the treatment of conditions such as psoriasis. EGCG exhibited kinetics characteristic of a slow, tight-binding inhibitor of 7,8-dihydrofolate reduction with bovine liver DHFR (K I = 0.109 Mmol/L), but of a classic, reversible, competitive inhibitor with chicken liver DHFR (K I = 10.3 M Amol/L). Structural modeling showed that EGCG can bind to human DHFR at the same site and in a similar orientation to that observed for some structurally characterized DHFR inhibitor complexes. The responses of lymphoma cells to EGCG and known antifolates were similar, that is, a dose-dependent inhibition of cell growth (IC 50 = 20 Mmol/L for EGCG), G 0 -G 1 phase arrest of the cell cycle, and induction of apoptosis. Folate depletion increased the sensitivity of these cell lines to antifolates and EGCG. These effects were attenuated by growing the cells in a medium containing hypoxanthine-thymidine, consistent with DHFR being the site of action for EGCG. (Cancer Res 2005; 65(6): 2059-64)