Pyridoxal phosphate as a constituent of the histaminase (benzylamine oxidase) of pig plasma

1. The transient kinetics of reduction of the 470-nm absorption band in benzylamine oxidase by substrate at different pH values between 6 and 10 have been studied by stopped-flow techniques, and substituent effects on kinetic parameters for the reduction process have been examined using a series of ring-substituted benzylamine derivatives as the substrates.2. Reduction of the enzyme by substrate takes place in two kinetically distinguishable steps, with the intermediate formation of an enzyme . substrate complex in which the substrate appears to be covalently bound through its amino group to the prosthetic group of the enzyme, possibly in the form of an amine-pyridoxal Schiff-base.3. The apparent stability of the enzyme . substrate complex shows no obvious dependence on the electronic properties of the amine substrates, but is strongly pH-dependent in a way suggesting that substrate-binding involves the non-protonated amines, exclusively, and requires the presence of the acid form of an ionizing group in the enzyme with apparent pK, of 8.8.4. Reduction of the enzymatic 470-nm chromophore and release of the aldehyde product of the catalytic process are rate-limited by the same monomolecular reaction step involving the enzyme . substrate complex. Rate constants for the rate-limiting reaction exhibit no significant dependence on pH between 6 and 10, but correlate with Hammett o-values for the ring-substituted benzylamine derivatives tested, yielding a @-value of + 0.3.Benzylamine oxidase, which catalyzes the oxidative deamination of various monoamines with formation of the corresponding aldehydes, hydrogen peroxide, and ammonia, contains copper and a prosthetic group thought to be pyridoxal phosphate [l-31. The free enzyme exhibits a 470-nm absorption band, which disappears on addition of amine substrates under anaerobic conditions and reappears on aeration of the enzyme solution [l]. Stopped-flow kinetic studies have shown that the transient reduction of this absorption band by substrate during enzyme turnover reflects a catalytically significant process, resulting in the formation of a reduced enzymatic intermediate [4-61. A recent investigation of the reactivity of benzylamine oxidase towards carbonyl reagents led to the prediction that forination of the reduced enzyme species, and hence catalytic activity, would be expected to be dependent upon the protonation state of the amine substrate and of an ionizing group in the enzyme with an apparent pK, of 8.8 [7]. This prediction has now been tested by a direct examination of the En-~rnr. Benzylamine oxidase or inonoamine : 0, oxidoreductase (deaminating) flavin-containing (EC 1.4.3.4). effect of pH on the transient reduction of the enzymatic 470-nm chromophore by benzylamine and some ringsubstituted benzylamine derivatives. The data presented below establish the kinetic significance of two separate reaction steps in the reduction process, and substituent and pH effects on each of these steps are desci-ibed and discussed. EXPERIMENTAL PROCEDURE MaterialsThe prepar...

Section: Discussionmentioning

confidence: 80%

Effect of pH on the Transient Reduction of Pig-Plasma Benzylamine Oxidase by Benzylamine Derivatives

Lindström¹,

Olsson²,

Olsson³

et al. 1976

“…Evidence has been presented indicating that the prosthetic group in this and related copper-containing amine oxidases is identical with, or closely related to, pyridoxal phosphate [1,2,19]. This idea has not been generally accepted, however, but alternative structures for the prosthetic group have been proposed.…”

Section: Discussionmentioning

confidence: 98%

“…Assuming that the production of hydrogen peroxide is associated with the oxygen-dependent reaction step, Eqn (1) prescribes that the amplitude (BHZ02) of the transient burst of product formation is given by where CE stands for the total concentration of enzyme and n denotes the number of active sites per enzyme molecule [16]. Eqn (2) shows that a full burst (a burst agreeing with the active-site concentration of enzyme) will be obtained when k 3 [ 0 2 ] 9 k4, i.e. when the oxygen-independent reaction in Eqn (1) is the main rate-limiting step in the process of enzyme reoxidation.…”

Section: Determination Of the Active-site Concentration Of Enzymementioning

confidence: 99%

“…The enzyme, which contains copper and a prosthetic group interacting strongly with carbonyl reagents [1,2], has been suggested to operate by a substitution (ping-pong) mechanism [3,4]. The interaction between enzyme and amine substrates thus appears to lead primarily to the formation of a reduced enzyme species, which is subsequently reoxidized by molecular oxygen [5 -71.…”

mentioning

confidence: 99%

See 1 more Smart Citation

The Order of Substrate Addition and Product Release during the Catalytic Action of Pig-Plasma Benzylamine Oxidase

Lindström¹,

Pettersson²

1978

Hydrogen peroxide is neither released, nor catalytically formed, during the anaerobic reduction of pig‐plasma benzylamine oxidase by the substrate benzylamine. This observation rules out the possibility that oxygen remains tightly bound (in a catalytically relevant manner) to the enzyme under standard experimental conditions of anaerobiosis, i.e. the possibility that benzylamine oxidase operates by a ternary‐complex mechanism is definitely excluded. During benzylamine oxidase catalysis in the presence of oxygen there is a transient burst of hydrogen peroxide release preceding the steady‐state phase of product formation. It can be inferred from the variation with pH and oxygen concentration of the rate and amplitude of this burst that hydrogen peroxide formation is associated with the oxygen‐dependent reaction step in the process of enzyme reoxidation. Approximately one mole of hydrogen peroxide per mole of enzyme is released in the burst observed at high pH and oxygen concentration, confirming that benzylamine oxidase contains a single catalytically active site. There is no production of ammonia from benzylamine during the catalytic action of benzylamine oxidase under anaerobic conditions. This observation rules out recently proposed mechanisms of enzyme action based on the presence of a sulfenic acid residue or a disulfide group functioning as an electron acceptor at the active site of the enzyme. It lends support to the original suggestion that benzylamine oxidase is a pyridoxal enzyme.

“…Although it is recognized that the reoxidation process may involve both oxygen-dependent and oxygen-independent reaction steps [ 5 ] , the above simplification is adequate for the present purpose, which is to discuss data referring mainly to the reduction process and obtained at a constant concentration of oxygen (aerobic conditions). Equation (2) predicts that the steady-state velocity of the catalytic reaction conforms to Michaelis-Menten kinetics with where…”

Section: Discussionmentioning

confidence: 99%

Kinetic Isotope Effects on the Catalytic Activity of Pig-Plasma Benzylamine Oxidase

Olsson¹,

Olsson²,

Pettersson³

1976

1. Isotope effects on the catalytic activity of benzylamine oxidase at pH 7 and 9 have been studied by steady-state and transient-state kinetics methods, using [a,a-2H]benzylamine as the substrate.2. Replacement of the a-hydrogen atoms in benzylamine by deuterium has no significant effect on substrate-binding to benzylamine oxidase, neither does it affect the rate of reoxidation of the reduced form of the enzyme. Conversion of the primarily formed enzyme . substrate complex into the reduced enzyme species, however, exhibits an isotope effect of about 3.3. The data obtained are consistent with a mechanism in which reduction of benzylamine oxidase takes place by a rapid pre-equilibration between enzyme and substrate to form an amine-pyridoxal Schiff-base, which is then tautomerized by a comparatively slow prototropic shift to an amino aldehyde-pyridoxamine Schiff-base from which there is a rapid hydrolytic release of the aldehyde product corresponding to the amine substrate. Proton abstraction from the a-carbon of the amine moiety in the primary Schiff-base appears to be at least partially rate-limiting for the tautomerization step, and hence for the entire process of enzyme reduction.Benzylamine oxidase, which catalyzes the oxidative deamination of various monoamines, contains copper and a prosthetic group thought to be pyridoxal phosphate [l-31. The enzyme appears to operate by a substitution (ping-pong) type of mechanism [4,5], in which the interaction between enzyme and an amine substrate primarily leads to formation of the corresponding aldehyde product and a reduced enzymatic intermediate [6,7]. The reduced enzyme species is subsequently reoxidized by oxygen in a process which is rate-limiting for the steady-state reaction under aerobic conditions [ 5 ] . Evidence has been presented indicating that reduction of benzylainine oxidase involves Schiff-base formation between enzyme-bound pyridoxal phosphate and the amine substrate [3,8], and the kinetic significance of a covalent enzyme . substrate complex possibly representing a Schiff-base intermediate has recently been established [9]. The detailed mechanism of the catalytic reaction, as well as the detailed nature of the enzymatic intermediates involved, however, remains obscure.