Lactate dehydrogenase: Electrophoretic behaviour electron microscopy and structure

The covalent binding of GSH and cysteine to mouse lactate dehydrogenase by disulphide bonds, gives rise to all but five bands of the complex electrophoretograms of the enzyme [Dudman, N.P.B. (1969) Biochem. Biophys. Res. Commun., 36, 608]. Other explanations have been proposed for this complexity, including that the enzyme exists as a number of conformational isomers [Houssais, J.-F. (1966) Biochim. Biophys. Acta 128, 239], and that the enzyme arises from the expression of three different genes [Costello, L. A. and Kaplan, N. O. (1963) Biochim. Biophys. Acta 73, 658]. The basis for these proposals is examined critically. From electrophoretograms of mouse lactate dehydrogenase both fully reduced, and covalently bound to GSH and cysteine, it is concluded that each muscle-type subunit has one especially active -SH group, but that the heart-type subunit has none. The state of ionization of the covalently bound GSH and cysteine residues, and of the free enzymatic thiol residues is also derived. Mouse lactate dehydrogenase is found to be adsorbed by some batches of polyacrylamide. Cathodic sub-bands of Isoenzyme 5 are bound most firmly, and adsorption appears strongest when the enzyme is fully reduced. Acrylamide gels containing pyruvate and NADH appear not to adsorb the enzyme. The possible effects of differing geometrical arrangements of subunits upon the electrophoretic pattern of lactate dehydrogenase are considered. Subunits of the enzyme from beef heart are found by electron microscopy to be arranged in flattened tetrahedra. This observation, taken with the electrophoretic pattern of the enzyme, indicates that beef lactate dehydrogenase comprises subunits of at least four distinguishable types

Jack bean urease: mixed-metal derivatives

We have prepared, for the first time, mixed-metal (Zn/Ni and Co/Ni) derivatives of the nickeloenzyme, urease. The mixed-metal derivatives, although possibly catalytically inert, provide a means for the further investigation of both the catalytic mechanism and coordination of the native nickel ions. A study of the specific activity of nickel-depleted urease indicates that the loss of one nickel ion is sufficient to render it inactive. Sulfite was found to play a key role in stabilising the urease-bound nickel. The visible spectrum of the pink Co/Ni urease is presented. Together, these results provide additional evidence for a difference in the ligation of the jack bean urease active-site nickel ions and comment on the possibility of a variety of ligation geometries. Examination of the sequences of the enzymes from Klebsiella aerogenes and the jack bean reveals that there is an exact match, residue for residue, between the critical ligating and active-site residues of the Klebsiella enzyme (α-chain residues: Asp 360, Cys 319, His 272, His 246, His 136, His 134 and carboxylated Lys 217) and those of the jack bean enzyme (Asp 633, Cys 592, His 545, His 519, His 409, His 407 and Lys 490), and this lends strong support to essentially identical mechanisms of action for the two enzymes

Ellman's reagent: 5,5′-dithiobis(2-nitrobenzoic acid)-a reexamination

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 reduction of commercial DTNB followed by gel chromatography 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 at 412 nm in dilute aqueous salt solutions. This value was confirmed independently by reduction of purified DTNB with cysteine. Titration of sulphydryl groups with DTNB can be done at pH 7.27 where the thiol group of TNB is 99.8% in the intensely-colored conjugate base form while the hydroxide-promoted hydrolysis of DTNB is minimal

On the mechanism of the reaction of tris(hydroxymethyl)aminomethane with activated carbonyl compounds: A model for the serine proteinases

The pH dependence of the reaction of tris(hydroxymethyl)aminomethane (Tris) with the activated carbonyl compound 4-trans-benzylidene-2-phenyloxazolin-5-one (I) is given by the equation k′ = kK (K + [H]) + k[OH]K (K + [H]), where K is the dissociation constant of TrisH. Spectrophotometric experiments show that the Tris ester of α-benzamido-trans-cinnamic acid is formed quantitatively over a range of pH values, regardless of the relative contribution of k and k terms to k′. Hence, both terms refer to alcoholysis. While the mechanism of the reaction is not determined unequivocally in the present work, the magnitude of the k term, together with its dependence on the basic form of Tris, suggests that ester formation is occurring by nucleophilic attack of a Tris hydroxyl group on the carbonyl carbon of the oxazolinone, with intramolecular catalysis by the Tris amino group. The rate enhancement due to this group is at least 10 and possibly of the order 10. This system is compared with other model systems for the acylation step of catalysis by serine esterases and proteinases

Specificity and pH Dependence of Ficin-Catalyzed Hydrolyses. Comparisons with Bromelain Specificity

The specificity of ficin has been investigated using a number of ester and peptide substrates. Three families of esters were used: (i) a series of hippuric acid esters, for which the identity of the values suggests the rate-determining deacylation of a hippuryl-ficin intermediate; (ii) a series of N-acylglycine p-nitrophenyl esters, for which k/K was found to be markedly dependent on the size of the nonpolar acyl group, increasing by a factor of 200 in going from the formyl to the trans-cinnamoyl derivative; and (iii) a series of N-benzyloxycarbonylamino acid p-nitrophenyl esters, in which the L-alanine and L-lysine derivatives were the best substrates examined. The effect of pH on k has been determined for the ficin-catalyzed hydrolyses of α-N-benzoyl-Larginine methyl ester and N-benzyloxycarbonyl-L-alanine pnitrophenyl ester. The effects of ficin and bromelain on a number of dipeptides, tripeptides, and polypeptides (bradykinin, angiotensin, and oxidized insulin A and B chains) have been studied. The results indicate that ficin and bromelain should prove generally useful in peptide sequencing, since they catalyze the hydrolysis of glycyl, alanyl, and leucyl bonds, as well as valyl, phenylalanyl, tyrosyl, and other bonds under more vigorous conditions