The mechanism of the reaction of horseradish peroxidase isoenzyme C (HRPC) with hydrogen peroxide to form the reactive enzyme intermediate compound I has been studied using electronic absorbance, rapid-scan stopped-flow, and electron paramagnetic resonance (EPR) spectroscopies at both acid and basic pH. The roles of the active site residues His42 and Arg38 in controlling heterolytic cleavage of the H(2)O(2) oxygen-oxygen bond have been probed with site-directed mutant enzymes His42 --> Leu (H42L), Arg38 --> Leu (R38L), and Arg38 --> Gly (R38G). The biphasic reaction kinetics of H42L with H(2)O(2) suggested the presence of an intermediate species and, at acid pH, a reversible second step, probably due to a neutral enzyme-H(2)O(2) complex and the ferric-peroxoanion-containing compound 0. EPR also indicated the formation of a protein radical situated more than approximately 10 A from the heme iron. The stoichiometry of the reaction of the H42L/H(2)O(2) reaction product and 2,2'-azinobis(3-ethylbenzothiazolinesulfonic acid) (ABTS) was concentration dependent and fell from a value of 2 to 1 above 0.7 mM ABTS. These data can be explained if H(2)O(2) undergoes homolytic cleavage in H42L. The apparent rate of compound I formation by H42L, while low, was pH independent in contrast to wild-type HRPC where the rate falls at acid pH, indicating the involvement of an ionizable group with pK(a) approximately 4. In R38L and R38G, the apparent pK(a) was shifted to approximately 8 but there is no evidence that homolytic cleavage of H(2)O(2) occurs. These data suggest that His42 acts initially as a proton acceptor (base catalyst) and then as a donor (acid catalyst) at neutral pH and predict the observed slower rate and lower efficiency of heterolytic cleavage observed at acid pH. Arg38 is influential in lowering the pK(a) of His42 and additionally in aligning H(2)O(2) in the active site, but it does not play a direct role in proton transfer.

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The mechanism-based inactivation of four horseradish peroxidase (HRP-C) cnzymc variants hits been studied kinetically with eithcr hydrogen peroxide o r the xcnobiotic m-chloropcroxybcnzoic acid (mC10,-BxOH) as sole substrate. The concentration and time dependcnce of inactivation was investigatcd for the wild-type plant enzyme (HRP-C), the unglycosylated rccombinant enzyme (HRP-C*), and two site-dirccted mutants with Phc143 replaced by Ala (I F143AIHRP-C:':) or Arg% replaced by Lys ([R3XK]HRP-P). The number of turnovers (r) of H,O, requircd to completely inactivate h e enzymes was found to vary between the different enzymes with HRP-C being iiiost resistant to inactivation ( u = 625), HRP-C* and IF1 43AIHRP-C* being approximately twice its sensitive ( r = 335 and 385, respectively) in coniparison, and LR38KIHRP-C* being inactivatcd much Inore casily ( r = 20). In the cases of HKP-C* and LF143A]HRP-C*, compared to HRP-C! the dirfcrences wcre duc to the iibsencc of glycosylation on the cxterior of the proteins, whilsl the [R3XKlHRP-(Y variant exhibited a distinct mechanistic difference. When mCI0,BAIH was U S G~ as the substratc the differences in sensitivity to inactivation disappeared. The valucs of r were all around 3 retlecting the strorig affinity of ~HCIO,BZOH for the active site. The apparent rate constant for inactivation by H 2 0 , was found to be about twofold higher in [R38K]HRP-C" than the other enxyincs and thc catalytic constant for turnover of H 2 0 1 wits approxirriately ten times lowcr.The al'finity of compound I for H,02 leading to the fortnation of a transitory interiiicdiate implicated in the inactivation of peroxidase dccreased in the order HRP-C, HRP-C", [F143A]HRP-C*, [R38K]HKP-C*.K6,ywordv: horseradish pcroxidase; recombinant; mutant; hydrogen peroxide; suicide inactivation.Horseradish peroxidase (HRP) (donor:hydrogen peroxide oxidorcductase) is a classical haem pcroxidase and a incinber ol' the supcrfaniily of peroxidase enzymes found in plants, fungi and bacteria. Following thc classification of Wclinder 11 J, HRP is a class IT1 secretory enzyme which is involvcd in the forrnation of free radicals vital in polymerisation reactions such as lignification and suberization. It occurs in it wide varicty of isoforms 121, slightly basic (cationic) HRP-C being the tnost aloun-The cnzyrrie has been characterised as a single polypeptide chain, heavily plycosylated ('I 8 % by mass) [4]

H2O2 is the usual oxidizing substrate of horseradish peroxidase C (HRP-C). In the absence in the reaction medium of a one-electron donor substrate, H2O2 is able to act as both oxidizing and reducing substrate. However, under these conditions the enzyme also undergoes a progressive loss of activity. There are several pathways that maintain the activity of the enzyme by recovering the ferric form, one of which is the decomposition of H2O2 to molecular oxygen in a similar way to the action of catalase. This production of oxygen has been kinetically characterized with a Clark-type electrode coupled to an oxygraph. HRP-C exhibits a weak catalase-like activity, the initial reaction rate of which is hyperbolically dependent on the H2O2 concentration, with values for K2 (affinity of the first intermediate, compound I, for H2O2) and k3 (apparent rate constant controlling catalase activity) of 4.0±;0.6mM and 1.78±;0.12s-1 respectively. Oxygen production by HRP-C is favoured at pH values greater than approx. 6.5; under similar conditions HRP-C is also much less sensitive to inactivation during incubations with H2O2. We therefore suggest that this pathway is a major protective mechanism of HRP-C against such inactivation.

H2O2 is the usual oxidizing substrate of horseradish peroxidase C (HRP-C). In the absence in the reaction medium of a one-electron donor substrate, H2O2 is able to act as both oxidizing and reducing substrate. However, under these conditions the enzyme also undergoes a progressive loss of activity. There are several pathways that maintain the activity of the enzyme by recovering the ferric form, one of which is the decomposition of H2O2 to molecular oxygen in a similar way to the action of catalase. This production of oxygen has been kinetically characterized with a Clark-type electrode coupled to an oxygraph. HRP-C exhibits a weak catalase-like activity, the initial reaction rate of which is hyperbolically dependent on the H2O2 concentration, with values for K(2) (affinity of the first intermediate, compound I, for H2O2) and k(3) (apparent rate constant controlling catalase activity) of 4.0 +/- 0.6 mM and 1.78 +/- 0.12 s(-1) respectively. Oxygen production by HRP-C is favoured at pH values greater than approx. 6.5; under similar conditions HRP-C is also much less sensitive to inactivation during incubations with H2O2. We therefore suggest that this pathway is a major protective mechanism of HRP-C against such inactivation.

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Dihydrofolate reductase (DHFR) is the subject of intensive investigation since it appears to be the primary target enzyme for "antifolate" drugs, such as methotrexate and trimethoprim. Fluorescence quenching and stopped-flow fluorimetry show that the ester bond-containing tea polyphenols (-)-epigallocatechin gallate (EGCG) and (-)-epicatechin gallate (ECG) are potent and specific inhibitors of DHFR with inhibition constants (K(I)) of 120 and 82 nM, respectively. Both tea compounds showed the characteristics of slow-binding inhibitors of bovine liver DHFR. In this work, we have determined a complete kinetic scheme to explain the slow-binding inhibition and the pH effects observed during the inhibition of bovine liver DHFR by these tea polyphenols. Experimental data, based on fluorimetric titrations, and transient phase and steady-state kinetic studies confirm that EGCG and ECG are competitive inhibitors with respect to 7,8-dihydrofolate, which bind preferentially to the free form of the enzyme. The origin of their slow-binding inhibition is proposed to be the formation of a slow dissociation ternary complex by the reaction of NADPH with the enzyme-inhibitor complex. The pH controls both the ionization of critical catalytic residues of the enzyme and the protonation state of the inhibitors. At acidic pH, EGCG and ECG are mainly present as protonated species, whereas near neutrality, they evolve toward deprotonated species due to ionization of the ester-bonded gallate moiety (pK = 7.8). Although DHFR exhibits different affinities for the protonated and deprotonated forms of EGCG and ECG, it appears that the ionization state of Glu-30 in DHFR is critical for its inhibition. The physiological implications of these pH dependencies are also discussed.