Gordon A. Hamilton

The valence of copper and the role of superoxide in the D-galactose oxidase catalyzed reaction

Abstract Evidence for the involvement of Cu(III) in an enzymic reaction (that catalyzed by D-galactose oxidase) is reported. Superoxide dismutase inhibits the rate of the D-galactose oxidase catalyzed reaction and causes a small increase in the EPR signal due to Cu(II). Both ferricyanide and superoxide activate the enzyme (frequently 4 fold or greater) and cause a decrease (to essentially zero in some cases) in the intensity of the EPR signal. These and other results suggest that in its catalytic cycle the enzyme oscillates between Cu(I) and Cu(III) with superoxide bound to a Cu(II) state being only a fleeting intermediate. The Cu(III) enzyme is apparently the oxidant which converts the primary alcohol function of galactose to an aldehyde.

The inhibition of mammalian d-amino acid oxidase by metabolites and drugs. Inferences concerning physiological function☆

Abstract The inhibition of d -amino acid oxidase (EC 1.4.3.3) by a large number of metabolites and drugs is reported. When the substrate is an adduct (thiazolidine-2-carboxylate) formed from cysteamine and glyoxylate, at least five different mechanisms of inhibition are possible, and examples of each are given. Effective inhibitors include (a) some adenosine containing nucleotides, in particular ADP, AMP, ADP ribose, NADH, NADPH and dephospho-CoA; (b) diuretics, especially furosemide, ethacrynic acid, and mersalyl; (c) anti-inflammatory agents, such as salicylate, indomethacin, phenylbutazone, acetylsalicylate, mefenamic, and flufenamic acids; (d) hypoglycemic, hypocalcemic, and hypolipidemic compounds, including 5-methylpyrazole-3-carboxylate, pyrrole-2-carboxylate, 5-methylthiophene-2-carboxylate, thiophene-2-carboxylate, benzoate, and nicotinate; (e) aldehydes, for example, formaldehyde, acetaldehyde, and succinate semialdehyde; (f) thiols, especially β-aminothiols such as cysteine and penicillamine; (g) tropolone, and (h) hydrogen peroxide. The rate of the reaction is also very sensitive to oxygen presure; at pH 7.4 and 25°C the K m for O 2 is 1.1 m M . These data, in conjunction with literature information concerning the biological affects of such compounds, are used to suggest possible physiological processes in which the d -amino acid oxidase reaction may be involved. Although such correlations based on circumstantial evidence are not conclusive, they suggest that d -amino acid oxidase may play a major role in the control of metabolism in animals. Some processes in which it may participate include maintenance of ion and water balance in the kidney, inflammatory response, transmission of nerve impulses, sensing of O 2 concentration, control of cell growth, and as part of an intracellular messenger system for some hormones, especially insulin.

Thiazolidine-2-carboxylate derivatives formed from glyoxylate and L-cysteine or L-cysteinylglycine as possible physiological substrates for D-aspartate oxidase.

Adducts of glyoxylate with L-cysteine or L-cysteinylglycine were found to be excellent substrates at low concentrations for beef kidney D-aspartate oxidase. Evidence is presented that cis-thiazolidine-2,4-dicarboxylate and its glycine amide are the actual substrates, and that both are converted in the enzymic reaction to 4-substituted thiazoline-2-carboxylates. The results imply that these thiazolidine derivatives are the likely physiological reactants for mammalian D-aspartate oxidase.

Equilibrium constants for the formation of glyoxylate thiohemiacetals and kinetic constants for their oxidation by O2 catalyzed by l-hydroxy acid oxidase

Abstract Glyoxylate thiohemiacetal formation constants (defined as the concentration of thiohemiacetal divided by the concentration of thiol and the total concentration of hydrated and unhydrated glyoxylate) were determined at 25°C and pH 7.4 for a variety of thiols using two independent methods, and were found to be in the range of 0.2 to 1.7 m m −1. Under the same conditions the hydration constant for glyoxylate (defined as the concentration of the hydrate divided by the concentration of the free aldehyde) was determined to be 163 ± 7. This information is used in conjunction with kinetic data to calculate kinetic constants for the oxidation of the thiohemiacetals by O2 catalyzed by rat kidney l -hydroxy acid oxidase. The results further indicate that several such thiohemiacetals are excellent substrates, and suggest that one or more of them may be the physiological reactant for this enzyme.

Thiol-glyoxylate adducts as substrates for rat kidney L-α-hydroxy acid oxidase

Abstract Rat kidney L-α-hydroxy acid oxidase (EC 1.1.3.15) catalyzes a rapid O2 uptake at pH 7.5 when both glyoxylate and one of a number of various thiols are present. Thiols which are reactive in this system include: ethanethiol, 1-propanethiol, 2-mercaptoethanol, N-acetylcysteamine, propane-1,3-dithiol, dihydrolipoic acid, Coenzyme A, dephospho Coenzyme A, phosphopantetheine, and pantetheine. Notable physiological thiols that are not very reactive include: glutathione, L-cysteine and cysteamine. Presumably the substrate is a thiol-glyoxylate adduct because both the thiol and glyoxylate must be present in order to obtain a rapid enzyme-catalyzed reaction and oxalyl thioesters are the products of the enzymic reactions. Kinetic studies indicate that some of these adducts are better substrates than any others presently known. These and other results imply that a thiol-glyoxylate adduct may be the physiological substrate for L-α-hydroxy acid oxidase. A possible function for this reaction in metabolic control, mediated either by the oxalyl thioester or by oxalate, is briefly considered.

Inhibition of dopamine β-hydroxylase by thiazoline-2-carboxylate, a suspected physiological product of D-amino acid oxidase

Abstract Thiazoline-2-carboxylate was chemically synthesized and shown to be identical in all respects to the product formed in a D-amino acid oxidase catalyzed reaction involving cysteamine and glyoxylate. Both the chemically synthesized and enzymically prepared thiazoline-2-carboxylate are effective inhibitors of dopamine β-hydroxylase but they do not appreciably affect the activity of several other metalloenzymes that require copper, iron or zinc. The inhibition of dopamine β-hydroxylase is competitive with respect to the reactant ascorbic acid and uncompetitive with respect to tyramine. The possible physiological significance of this inhibition is briefly considered.

An oxygen-18 tracer investigation of the mechanism of myo-inositol oxygenase

Abstract In the conversion of myo -inositol to D-glucuronic acid catalyzed by myo -inositol oxygenase only one atom of 18O from 18O2 is incorporated into the product, and it is found exclusively in the carboxyl group. Control experiments indicate that under the reaction conditions no exchange of solvent oxygens with D-glucuronate occurs. To avoid exchange during isolation and analysis the oxygenase product was enzymically reduced to L-gulonate and isolated in that form. The results eliminate one possible mechanism for the oxygenase reaction, but are consistent with two others which seem chemically reasonable.

A reactive aspartyl residue of pepsin.

Abstract Recently it was reported ( Hamilton, Spona, and Crowell, 1967 ) that pepsin was inactivated by an equimolar amount of 1-diazo-4-phenylbutanone-2 (DPB). The results indicated that the reaction occurred at or near the active site of pepsin. In the present communication we report evidence indicating that DPB reacts with pepsin to form an ester of 1-hydroxy-4-phenylbutanone-2 (HPB) with the β-carboxyl group of an aspartyl residue, and that the amino acid sequence containing this aspartyl residue is: Ile-Val-Asp-Thr. This aspartyl residue is different from that attacked by another class of pepsin inhibitors, the α-haloketones ( Erlanger, Vratsanos, Wassermann, and Cooper, 1966 ). Therefore, the present results define a different section of the pepsin molecule which may be involved in the enzymic catalysis.

Purification and properties of hog liver 4-hydroxyphenylpyruvate dioxygenase.

Abstract The purification of hog liver 4-hydroxyphenylpyruvate dioxygenase (EC 1.13.11.27), and the determination of some of its characteristics, are reported. The enzyme was purified 330-fold in 22% yield from an acetone powder extract by ammonium sulfate fractionation, chromatography twice using sulfopropyl Sephadex under carefully controlled pH conditions (once at pH 5.36 and a second time with a pH gradient from 5.25 to 5.80), and a final chromatography on DEAE-cellulose. The purified enzyme was found to be homogeneous by several standard criteria, but activity measurements indicated that a small amount (less than 5%) of a carboxylesterase (EC 3.1.1.1) isoenzyme is present as a minor impurity. On long-term storage at −20 °C the enzyme forms polymers but this can be reversed with thiols. The molecular weight of the freshly prepared or depolymerized enzyme was estimated to be 89,000 ± 2000 by equilibrium ultracentrifugation, and 50,000 to 54,000 by gel filtration. Sodium dodecyl sulfate-gel electrophoresis experiments, performed in the presence and absence of mercaptoethanol, indicate that the enzyme is composed of two nonidentical subunits with similar molecular weights (44,000 ± 2000). The enzyme gives a typical protein ultraviolet absorption spectrum with no noticeable peaks above 300 nm, it has no detectable carbohydrate content, and it contains 0.9 atom iron and 0.4 atom copper/89,000 daltons. Added iron and copper salts activate the enzyme to some extent but by less than a factor of 2. The enzymatic reaction has a large temperature coefficient (the rate increases ca. fivefold for each 10 °C rise) and is markedly stimulated (up to sixfold) by the presence of some organic solvents in concentrations up to 10% of the medium. These results suggest that a protein conformation change, possibly aided by binding of the organic solvent, is involved in the rate-determining step of the reaction. The similarities and differences of this 4-hydroxyphenylpyruvate dioxygenase to those from other sources, and to prolyl hydroxylase, are discussed.

The nonenzymic hydrolysis of Δ2-thiazoline-2-carboxylate: The product of the suspected physiological reaction catalyzed by d-amino acid oxidase

Abstract Δ 2 -Thiazoline-2-carboxylate, the product of the suspected physiological reaction catalyzed by d -amino acid oxidase, is stable to hydrolysis at 37°C and pH 7 or above, but it hydrolyzes readily at pH 5 or below to give a mixture of N - and S -oxalylcysteamines; the N -oxalyl derivative predominates at pHs above 1 while the S -oxyalyl compound is the major product at high acidities. The pH-rate profile looks like the superposition of two bell-shaped curves. The initial increase in the rate as the pH is lowered is controlled by a p K a of 3.95 and from pH 1 to 3 the rate is relatively constant ( k = 6.7 × 10 −4 s −1 at 37°C and ionic strength 0.5 m ). Below pH 1 the rate increases again to a maximum in 1 m HCl and then decreases in more highly acidic solutions. The rate of conversion of S -oxalylcysteamine to N -oxalylcysteamine is inversely proportional to the hydrogen ion concentration from pH 3 to 5 but becomes largely independent of pH from pH 1 to 2. In the pH-independent region the rate is comparable with that observed by others for S -acetylcysteamine but in the pH-dependent region the rate is 20 to 25 times faster for the oxalyl derivative than for the acetyl compound. At pH 1, N -oxalylcysteamine is partially converted to the S -oxalyl derivative but the rate of hydrolysis ( k = 1.0 × 10 −5 s −1 at 37°C) to cysteamine and oxalate of this partially equilibrated system occurs at a comparable rate. The results of this investigation are rationalized in terms of what is known about other thiazoline hydrolyses and intramolecular S to N acyl migrations. The main differences in the present case are presumably due to the fact that thiazoline-2-carboxylate can undergo hydrolysis by two reaction manifolds, one with the carboxyl unprotonated and the other with it protonated. The relevance of these results to possible reactions of thiazoline-2-carboxylate in vivo is briefly considered.