The mechanism of catalase action. I. Steady-state analysis

1.

1. Tritium-labeled cholic acid was prepared by biosynthesis in the rat.     

Kinetics and mechanism of catalase action. Formation of the intermediate complex

The kinetics of formation of the intermediate complex between catalase and H₂O₂ has been reexamined. It has been shown that the kinetics consists of a rapid and of a subsequent slow phase. At the maximum of the transient decrement of the optical absorption, the system was found to be in a terminal state with regard to the rapid phase. On this basis, the formation curve of the intermediate complex was calculated. From the parameters of the curve the maximal saturation of catalase hematins (from horse erythrocytes) by H₂O₂ is 35%. The absolute spectrum of the intermediate complex was established. The variation of the previously calculated rate constant of formation of the intermediate complex was shown to be due to the inapplicability of the pre-steady-state approximation to the rate data. By applying a more general approach and by the use of a computer, the individual rate constants of the peroxidatic scheme were calculated (relevant to micromolar solutions of catalase) k₁ = (3.0 ± 0.2) × 10⁶ M^?1 sec^?1k₄ = (5.6 ± 0.3) × 10⁶ M^?1 sec^?1 These values are 2.2 times higher in a nanomolar solution.     

A proposed mechanism for the catalatic action of catalase

A detailed mechanism for catalatic action has been proposed which includes the formation of Chance's catalase compound I in the first step and hydride ion transfer in the second step. The first (oxidative) step involves direct reaction of hematin iron with an ionized H2O2 molecule, followed by an oxidation of the iron to Fe IV. The second step is assumed to depend upon the reductive action of a second H2O2 molecule on Chance's compound I through a catalyzed hybride ion transfer, resulting in the regeneration of uncomplexed catalase. Differences between the catalatic and peroxidative actions of catalase are discussed briefly in respect to the proposed mechanism for catalatic action. The rationale of the proposed mechanism is based to a considerable extent upon the type of ligand binding by the hematin iron of catalase, and this type of ligand bonding is contrasted with ligand binding in methemoglobin, which does not show catalatic activity. Finally, the dispositions of electrons in the outer electronic orbitals of the hematin iron of catalase and methemoglobin are discussed, as a means of justifying formulae presented for catalase and methemoglobin and their derivatives. One of the features of the proposed catalatic mechanism is the assumption, based on electron spin number, that the sixth coordination position around the hematin iron of uncomplexed catalase is unoccupied.     

A mechanism for NADPH inhibition of catalase compound II formation.

Catalase-bound NADPH both prevents and reverses the accumulation of inactive bovine liver catalase peroxide compound II generated by 'endogenous' donors under conditions of steady H2O2 formation without reacting rapidly with either compound I or compound II. It thus differs both from classical 2-electron donors of the ethanol type, and from 1-electron donors of the ferrocyanide/phenol type. NADPH also inhibits compound II formation induced by the exogenous one-electron donor ferrocyanide. A catalase reaction scheme is proposed in which the initial formation of compound II from compound I involves production of a neighbouring radical species. NADPH blocks the final formation of stable compound II by reacting as a 2-electron donor to compound II and to this free radical. The proposed behaviour resembles that of labile free radicals formed in cytochrome c peroxidase and myoglobin. Such radical migration patterns within haem enzymes are increasingly common motifs.     

Kinetic studies on the mechanism of pepsin action.

The linear noncompetitive inhibition of the pepsin-catalyzed hydrolysis of Ac-Phe-Phe-Gly at pH 2.1 by L-Ac-Phe, L-Ac-Phe-NH2, and L-Ac-Phe-OEt has been claimed to substantiate the ordered release of products specified by the amino-enzyme mechanism for pepsin action. According to this interpretation, the binding of inhibitor to free enzyme and the amino-enzyme intermediate (Scheme I) generates the observed inhibition pattern. The proposition is valid only if a simple alternative explanation for the kinetic data, Scheme II, can be disproved. Scheme II attributes the inhibition pattern to the binding of inhibitor to free enzyme and the enzyme-substrate (Michaelis) complex. The experiments reported here have enabled us to distinguish between the two mechanisms. The pepsin-catalyzed hydrolyses of Ac-Phe-Trp, Z-H'IS-Phe-Trp, Z-Gly-His-Phe-Trp, and Z-Ala-His-Phe-Trp at pH 1.8 occur exclusively at the Phe-Trp bond and must yield the same amino-enzyme, E-Trp, if it is implicated. Under these circumstances, Scheme I requires that a plot of 1/kc vs. (I)o for the four substrates and a given noncompetitive inhibitor provide a set of four parallel lines. Scheme II predicts that the four lines generally will not be parallel. L-Ac-Phe, L-Ac-Phe-NH2, L-Ac-Phe-OMe, and D-Ac-Phe act as linear noncompetitive inhibitors for the pepsin-catalyzed hydrolysis of the four Trp-containing substrates. The plot of 1/kc vs. (I)o for each inhibitor results in a set of four nonparallel lines. Therefore Scheme II must be correct and the detection of noncompetitive inhibition accompanying the pepsin-catalyzed hydrolysis of peptides offers no insight into the merits of the amino-enzyme hypothesis.     

β-endorphin release by angiotensin II: studies on the mechanism of action

Blood-borne angiotensin II induces release of β-endorphin-like immunoreactivity (β-EI) from rat anterior pituitary gland. To study the mechanism of action we investigated in rats the effect of transection of subfornical organ efferent projections on angiotensin-induced β-EI release in vivo and also the direct action of angiotensin II on β-EI release from isolated adenohypophyses in vitro. (i) No effect of transection of subfornical organ efferents on the increase in plasma β-EI following intravenous infusions of angiotensin II was found. (ii) When anterior pituitary quarters were continuously superfused in vitro, angiotensin II (1 – 10 nM) caused release of β-EI into the superfusion medium in a dose-dependent manner. The stimulatory effect of angiotensin II (3 nM) was blocked by the receptor antagonist saralasin (300 nM). We conclude that β-endorphin release by blood-borne angiotensin II, in contrast to other central effects of angiotensin, is not mediated by the subfornical organ; instead a direct action of angiotensin II on the adenohypophysis could be a mechanism of action responsible.     

The action of ultraviolet radiation on yeast catalase

The effect of prolonged UV irradiation (mostly 2537 A) on the catalase activity of an aqueous yeast suspension was divisible into 4 periods. First, the period during which the cells lost their ability to form colonies, but during which no change in catalase activity was noted. Second, the period during which a considerable rise in catalase activity (Euler effect) occurred. The Euler effect was accompanied by enzyme alteration as shown by the simultaneous decrease in the activation energy of the enzyme-substrate system. However, during the initial phase of this period, as the catalase activity of the suspension began to increase, the activation energy rose to a transient level higher even than that characterizing the unaltered enzyme. Heat accelerated the rate of alteration when applied either during or after the irradiation; the activation energy for the over-all alteration reaction was 24 kcal., a value close to that recorded previously for alteration induced by chemical agents. Nevertheless, the rate-limiting step appeared to be different in the two cases. A model of these events was presented in which the primary photochemical action was on the site at which catalase is located within the cell. Third, a rather long period during which irradiation led to no diminution in the catalase activity of the maximally active suspension. This protection effect was duplicated in intro by a model crystalline catalase-KNA system, or by adding either ribonuclease digestion products of RNA or adenine to a catalase solution prior to irradiation. Evidence was adduced that the protection effect was not a simple screening, but involved some sort of interaction between the enzyme and the nitrogenous components of RNA, an interaction which must likewise occur within the cell. Alteration induced by CHCl₃ did not eliminate the protection effect, but that by butanol did. The onset of photoinactivation was due to modification of protein structure, not of RNA. Fourth, the period of photoinactivation of the intracellular enzyme, which was quite similar to that of the crystalline enzyme in vitro.     

Spectral studies of human erythrocyte catalase.

The optical absorption and circular dichroic spectra of human erythrocyte catalase (EC 1.11.1.6) and its cyanide, azide, and fluoride derivatives over the wavelength range of 210 to 700 nm are reported. Treatment with acid or alkaline solutions causes spectral changes which may be due to dissociation of the enzyme into subunits and removal of the heme group from the protein. The fractions of the protein structure present as alpha helix, beta pleated sheet, and unordered structure have been estimated from the CD spectrum in the far-ultraviolet region. The CD spectra also indicate that the protein conformation does not change appreciably after cyanide binding. The epr spectroscopy of the native enzyme and its cyanide complex are reported. The spectral results are compared with catalase obtained from other mammalian and bacterial sources.