Reverse Ordered Sequential Mechanism for Lactoperoxidase with Inhibition by Hydrogen Peroxide

Lactoperoxidase (LPO, FeIII in its resting state in the absence of substrates)—an enzyme secreted from human mammary, salivary, and other mucosal glands—catalyzes the oxidation of thiocyanate (SCN−) by hydrogen peroxide (H2O2) to produce hypothiocyanite (OSCN−), which functions as an antimicrobial agent. The accepted catalytic mechanism, called the halogen cycle, comprises a two-electron oxidation of LPO by H2O2 to produce oxoiron(IV) radicals, followed by O-atom transfer to SCN−. However, the mechanism does not explain biphasic kinetics and inhibition by H2O2 at low concentration of reducing substrate, conditions that may be biologically relevant. We propose an ordered sequential mechanism in which the order of substrate binding is reversed, first SCN− and then H2O2. The sequence of substrate binding that is described by the halogen cycle mechanism is actually inhibitory.

Enzymatic sulfation of steroids. XI. The extensive purification and some properties of hepatic sulfotransferase III from female rats

Our earlier studies showed that livers from female rats contained three glucocorticoid sulfating enzymes we named sulfotransferases I, II, and III, (STI, STII, and STIII, respectively). In this report STIII from female Charles River CD rats was purified 1010- to 1300-fold compared with liver homogenates. The most highly purified STIII fraction electrophoresed as a single protein band. The molecular weight of STIII was 68 300 ± 4900. Its pH optimum for cortisol sulfation was pH 6.0 ± 0.1. However, it was routinely assayed at pH 6.8 for reasons enumerated in the text. Cortisol sulfation by STIII proceeded by either an ordered sequential mechanism or by an Iso Theorell-Chance mechanism at pH 6.8. The Km's for cortisol and the reaction coenzyme, 3′-phosphoadenosine-5′-phosphosulfate were 6.48 ± 0.78 and 6.78 ± 1.26 μM, respectively. Comparison of the ability of the enzyme to sulfate 40 μM cortisol, estradiol-17β, testosterone, deoxycorticosterone, and dehydroepiandrosterone, showed that the glucocorticoid was sulfated preferentially. Interestingly, its cortisol sulfotransferase activity was inhibited by a number of steroids. p-Hydroxymercuribenzoate inhibition studies indicated the presence of essential sulfhydryl groups in STIII. The enzyme was activated by divalent Ba, Ca, Co, Cr, Mg, Mn, and Ni salts. It was inactivated by Zn2+ and Cd2+ salts. The text compares STIII from female rats with other steroid sulfotransferases including the major glucocorticoid sulfotransferase from male rats.

Kinetic studies with skeletal-muscle hexokinase

Rat skeletal-muscle hexokinase was partially purified by ammonium sulphate fractionation and gel filtration. The mechanism of the skeletal-muscle hexokinase was studied kinetically by initial-velocity analysis and product inhibition. Glucose 6-phosphate was a non-competitive inhibitor of glucose and ATP. ADP was a non-competitive inhibitor of glucose and a competitive inhibitor of ATP. The data on product inhibition and initial-velocity analysis of skeletal-muscle hexokinase support an ordered sequential mechanism (ordered Bi Bi) where the addition of substrates and release of products is in the order: ATP, glucose, glucose 6-phosphate and ADP.

INTRACELLULAR DISTRIBUTION AND SOME PROPERTIES OF HEXOKINASE OF BOVINE ADRENAL MEDULLA

The intracellular distribution of the hexokinase of the adrenal medulla was studied. Most of the hexokinase was found, in about equal amounts, in the mitochondria and the nonparticulate cytoplasm. The amount of hexokinase associated with the mitochondria was shown to be dependent on the hexokinase concentration in the medium surrounding the mitochondria.The hexokinase of the adrenal medulla was shown to be noncompetitively inhibited by adenosine diphosphate (ADP) with respect to glucose and adenosine triphosphate (ATP). It was competitively inhibited with respect to glucose by mannose, fructose, and galactose. Glucose-6-phosphate (0.09 M) did not inhibit the hexokinase. The data on product inhibition and initial velocity analysis support the concept of an ordered sequential mechanism for adrenal-medulla hexokinase.