Bruce Levenberg

A Bacterial Pterin Deaminase

Publisher Summary This chapter describes the bacterial pterin deaminase. In the standard assay, the mixture contains 0.05 ml of pterin carboxylic acid, 0.8 ml of phosphate buffer, and 0.02 to 0.15 ml of enzyme in a final volume of 1.0 ml. Incubation is carried out at 23° in a 1-ml Beckman quartz cuvette with a light path of 1.00 cm. Reaction is begun by the addition of enzyme, and readings of optical density are taken every 3 minutes thereafter. The control cuvette contains buffer and enzyme, but no pterin carboxylic acid. With crude preparations light measurement at 360 mμ is employed. However, with more purified fractions of the deaminase, it is possible to follow the course of the reaction at 290 mμ, the wavelength at which there is observed the largest AE value between substrate and product. The most satisfactory conditions for storage of enzyme fractions were found to be at pH 9.0 in 0.1 M Tris buffer at 0°. Under such conditions pterin deaminase activity remained almost unchanged for several weeks. The pH optimum for pterin deaminase activity occurs between pH 6.3 and 6.7. Although the reaction proceeds in the complete absence of phosphate, the rate of deamination is slightly faster in phosphate than in Tris buffer.

[37a] ATP: Urea amidolyase (ADP) (Candida utilis)☆

Publisher Summary This chapter describes the assay, purification, and properties of urea amidolyase. Urea serves as the sole nitrogen source for the growth of certain yeasts and unicellular green algae that contain no detectable urease activity. Such cells possess urea amidolyase, which catalyzes Mg 2+ - and K + -dependent cleavage of a mole of urea to two moles of ammonia and one mole of carbon dioxide, concomitant with the breakdown of a mole of adenosine triphosphate (ATP) to adenosine diphosphate (ADP) and inorganic phosphate. The formation of urea amidolyase is repressed by ammonia. Two types of assays are recognized for routine use. The first involves the measurement of the rate of release of 14 CO 2 from urea- 14 C and offers the advantage of convenience and high sensitivity. This assay is, particularly useful with crude extracts, because it is largely unaffected by the presence of contaminating enzymes, such as myokinase, ATPase, and 2,4-Dinitrophenylhydrazine (DPNH) oxidase. The second method is based on a coupled spectrophotometric determination of ADP using phosphoenolpyruvate, pyruvate kinase, and lactate dehydrogenase. The decrease in absorbance at 340 m μ , resulting from the oxidation of DPNH, which accompanies the conversion of pyruvate to lactate, allows a continuous determination of the rate of cleavage of ATP to ADP. This method is limited to purified amidolyase preparations, because cruder fractions often contain high levels of ATPase and other interfering activities. In Tris buffers, the optimum pH for amidolyase activity (assay method 1) is 7.8–7.9.

l-3-(3-carboxyfuran-4-yl)alanine, a new amino acid from the mushroom Phyllotopsis nidulans☆

Abstract A new amino acid has been discovered in uncombined form in extracts of the fruiting bodies of the mushroom, Phyllotopsis nidulans . Chemical and spectroscopic data support formulation of the structure as l -3-(3-carboxyfuran-4-yl)alanine.

Separation and differential sensitivity toward avidin of carbamyl phosphate synthetase and urea amidolyase

This enzyme is remarkably sensitive to inhibition by highly purified egg white avidin, a phenomenon which can be completely prevented by inclusion of excess biotin in the assay system. Inasmuch as avidin has been found to selectively inhibit all known biotinenzymes and, indeed, is now accepted as a diagnostic tool for the detection of such reactions [2], the proposal was made that UALase belongs to that class of enzymes which contain biotin in the form of a bound, functionally-active prosthetic group. A recent publication by Wellner, Santos and Meister [3], asserting that the glutamine-dependent carbamyl phosphate synthetase (CPSase, EC 2.7.2.5) of Escherichia coli may also be a biotinznzyme, has focused our attention on the relevant question of whether these two enzymic processes might possibly represent analogous activities of the same protein molecule. At this time we wish to report results which indicate that not only is each reaction catalyzed by a

Urea amidolyase of Candida utilis: Characterization of the urea cleavage reactions

Evidence is presented that the enzymes catalyzing the three reactions involved in urea cleavage in Candida utilis, biotin carboxylation, urea carboxylation, and allophanate hydrolysis occur as a complex of enzymes. The allophanate-hydrolyzing activity could not be separated from the urea-cleaving activity using common methods of protein purification. Further, urea cleavage and allophanate hydrolysis activities are induced coordinately in cells grown on various nitrogen sources. The reactions involved in urea cleavage can be distinguished from one another on the basis of their sensitivities to (a) heat, (b) pH, and (c) chemical inhibitors. Evidence is presented for the product of the first reaction in urea cleavage, biotin carboxylation. Production of carboxylated enzyme is ATP dependent and avidin sensitive. Carboxylated enzyme is not observed in the presence of 1 mM urea.

[23] Glutamine-Dependent carbamyl-P synthetase (Agaricus bisporus)☆

Publisher Summary This chapter discusses the assay, purification, and properties of glutamine-dependent carbamyl-P synthetase. Enzyme preparations are essentially obtained and partially purified from the sporophore extracts of Agaricus bisporus. In the presence of an excess of ornithine and ornithine transcarbamylase, 14C-labeled carbamyl-P, synthesized from H14CO-3 and the other necessary components of the system, is converted quantitatively to the acid-stable compound, citrulline. Under these conditions, activity is determined by measuring the rate of fixation of radioactivity from KH14CO3 into a form that remains nonvolatile in HCl. An almost absolute dependency on ornithine can be obtained even in crude, dialyzed extracts provided they are passed through a small column of Dowex 50 resin at 3° just prior to being assayed. Also, the divalent metal (M)-ion requirement of the reaction is less specific. In potassium phosphate buffer, with glutamine as nitrogen donor, the optimum pH for the coupled assay is in the range 7.4–7.9. On increasing the ammonium ion concentration and the pH of the incubation medium, a definite, but relatively low, activity may be seen for the ion in replacing glutamine as nitrogen donor.