Accurate determination of thiol groups by means of Ellman's reagent [5,5'-dithiobis(2-nitrobenzoic acid), DTNB] has been limited by uncertainty about the molar absorption coefficient of the dianion of the product, 2-nitro-5-thiobenzoic acid (TNB). A procedure is described for the purification of TNB by the reduction of commercial DTNB followed by gel filtration and crystallization. Pure DTNB is prepared by reoxidation of purified TNB followed by gel chromatography and crystallization. The molar absorption coefficient of the dianion of TNB is 14,150 M-1cm-1 at 412 nm in dilute aqueous salt solutions. This value has been confirmed independently by reduction of purified DTNB with cysteine. The conditions for use of DTNB have been examined and discussed in detail. Titration of sulphydryl groups can be done at pH 7.27 where the thiol group of TNB is 99.8% in the intensely-coloured conjugate base form, while the hydroxide-promoted hydrolysis of DTNB is minimal. The reaction of aromatic disulphides with hydroxide ion is discussed in terms of available data from the literature. It is argued that an intermediate (3-carboxylate-4-nitrophenylsulphenic acid) should be experimentally observable when DTNB reacts with highly concentrated hydroxide ion. Preliminary evidence for the presence of the sulphenic acid is presented.
The molecular weight of native urease is shown to be ~560,000 daltons by gel chromatography. The subunit molecular weight has been established from SDS-polyacrylamide gel electrophoresis to be ~91,000 daltons which is consistent with estimations of the equivalent weight using titrations with cinnamohydroxamate. An accurate determination of the total cysteine content (14.8 residues per 97,000 dalton subunit) has been carried out using DTNB as titrating agent under carefully determined conditions. It is demonstrated that a 97,000 dalton subunit contains 5.0 tryptophan residues Several procedures which have the potential for the reversible removal of the active-site nickel ion(s) have been investigated, but in all cases active urease could not be regenerated.
The substrate specificity of jack bean urease has been investigated using enzyme solutions of known normality. The enzymatic hydrolysis of formamide is confirmed using a continuous assay system for measuring ammonia. The values of kcat and Km are 85 s-1 and 1.09 M at pH 7.00, and 209 s-1 and 0.875 M at pH 5.21. The urease-catalyzed hydrolysis is competitively inhibited by fluoride giving values for Ki of 1.32 mM and 8.21 µM at pH 7.00 and pH 5.21 respectively. Two new substrates for urease, acetamide and N-methylurea, have been discovered using the continuous ammonia assay and have values for kcat and Km at pH 7.00 of 0.55 s-1 and 0.75 M (acetamide), and 0.075 s-1 and 0.221 M (N-methylurea). The Ki for the competitive inhibition by fluoride ion is 2.21 mM (acetamide) and 2.24 mM (N-methylurea). It is demonstrated that the various substrates for urease are hydrolyzed at a common active site which involves nickel ion(s). Enzymatic hydrolysis of the following compounds has not been detected: trifluoroacetamide, p-nitrophenyl carbamate, thiourea and O-methylisouronium ion.
A mechanism of action for jack bean urease is proposed and is discussed in terms of the essential components of the active site, and its consistency with the extensive available data. The proposed mechanism accounts satisfactorily for the substrate specificity of urease, the variation in pH-rate profiles among different substrates, and the failure of methanol to trap a carbamoyl- or carbamato-enzyme intermediate in the hydrolysis of urea. The proposed mechanism allows for predictions which can be experimentally tested.
The evidence for the formation of an acyl-enzyme in the α-chymotrypsin-catalyzed hydrolysis of substrates is briefly described. The formation of only one intermediate (other than the enzyme-substrate complex) is sufficient to account for the wealth of data available in the literature. A recent paper [Chibber, B.A.K., Tomich, J.M., Mertz, E.F. and Viswanatha, T. (1977) Proc. Natl. Acad, Sci, USA 74, 510-514] presented evidence which was taken to support the existence of an intermediate in the deacetylation of acetyl-α-chymotrypsin. This work has been reexamined, and the results negate the existence of the proposed additional acetyl-enzyme intermediate.
A 'simple' procedure for the preparation of bis(acetic-1-14C) anhydride and bis(butyric-1-14C) anhydride from the labelled sodium salts and the unlabelled anhydrides is described. The conditions produce ~100% exchange, and the products are obtained in high to moderate yield. The method is potentially useful for the synthesis of a wide range of carboxylic anhydrides.
LIST OF PUBLICATIONS
Riddles, P.W., de Jersey, J. and Zerner, B. (1978) Absence of evidence for an intermediate in the deacetylation of acetylchymotrypsin, Proc. Natl. Acad. Sci. USA 75(1), 172-174.
Riddles, P.W., Blakeley, R.L. and Zerner, B. (1979) Anal. Biochem, 74, 75-81. doi:10.1016/0003-2697(79)90792-9
Dixon, N.E., Gazzola, C., Riddles, P.W., Blakeley, R.L. and Zerner, B. (1979) Jack Bean Urease (EC S.5.1.5). On the Action of Urease on Urea, Formamide, Acetamide, N-Methylurea and Related Compounds, Submitted to Can. J. Biochem. , paper 6 DOI:10.1139/o80-181
Dixon, N.E., Riddles, P.W., Blakeley, R.L. and Zerner, B. (1979) Towards A Mechanism of Action of Jack Bean Urease (EC 3.5.1,5), Submitted to Can. J. Biochem., paper 7.
Blakeley, R.L., Riddles, P.W. and Zerner, B. (1979) Variable Stoichiometry in the Decomposition of Aromatic Disulfides in Alkaline Solution. On the Properties of 3-Carboxylate-4-nitro-benzenesulfenate ion. Submitted to Phosphorus and Sulfur. DOI:10.1080/03086648008078231