P-Mercuribenzoate

The fact that ATP and CTP bind to the same site follows from the observation that adding ATP to the inhibited enzyme by CTP reduces or reverses the inhibition, presumably because ATP competes with CTP for the same site. The fact that CTP binds to an allosteric site (i.e., it is not a competitive inhibitor) follows from the so-called desensitization effect. Addition of mercurials [e.g., p-mercuribenzoate (PMB)] reduces or eliminates the inhibition by CTP. However, it has no effect on the enzymatic activity of ATCase, presumably because the mercurials affect the regulatory subunits but not the catalytic site. As for the mechanism of cooperativity (both positive and negative), it is known that CTP does induce changes in the quaternary structure of the enzyme. 

Carbonic anhydrase activities of the egg shells are inhibited by reaction with p-mercuribenzoate by blocking reactive SH-groups of the enzyme. Omission of 2-mercaptoethanol from buffer solutions for extraction and fractionation also resul-... 

The molecular weight of 320,000 obtained for the muscle enzyme from sedimentation-diffusion data at 2-6 mg/ml and v = 0.75 (132) is to be compared with 270,000 obtained by Wolfenden et al. from s20,w = 11.1 S and D2 ,w = 3.75 X 10 7 cm2 sec1, and v = 0.731 calculated from the amino acid content (92). The rabbit muscle enzyme has a normal amino acid content, that is, no unusually low or large amount of a particular amino acid was found. Of the 32 cysteine/half-cystine residues per mole based on a molecular weight of 270,000, 6.2 were rapidly titrated with p-mercuribenzoate (92). Typical protein absorption spectra were reported for elasmobranch fish (126), carp (125), rat (127), and rabbit muscle enzyme (68). An E m at 280 nm = 9.13 has been reported for the rabbit muscle enzyme (133). The atypical absorption spectrum with a maximum at 275-276 nm observed by Lee (132) is indicative of contaminating bound nucleotides. 

The two preparations from A. oryzae reportedly differ in amino acid and carbohydrate composition. The enzyme prepared by Minato contained 25% carbohydrate no cysteine was detected either by titration with p-mercuribenzoate in 6M urea or by cysteic acid analysis after performic acid oxidation (179). In contrast, Wolfenden et al. (92) reported 14 cysteine residues per mole of enzyme which reacted instantaneously with p-mercuribenzoate in the absence of urea. No explanation is available for this apparent discrepancy. 

Prostatic acid phosphatase is reversibly inactivated by p-mercuri-benzoate and by Cu2+ and Fe3+ (59). In contrast to red cell acid phosphatase, prostatic acid phosphatase is only partially inactivated even after prolonged periods of incubation at high concentrations of p-mercuri-benzoate. Addition of cysteine to the p-mercuribenzoate-treated enzyme produces complete reactivation. Binding of SH groups by p-mercuri-benzoate renders the enzyme more labile to thermal denaturation, but no difference is obtained with surface inactivation (23). Similar partial inactivation with Cu2+ is also subject to reactivation. 

Other sulfhydryl reagents, such as p-mercuribenzoate and iodoaceta-mide, produced similar activation (44), except that with these compounds increases in activity were also observed at pH 9.1 (Table I). With p-mercuribenzoate maximum activation was observed when 2-4 sulfhydryl groups were titrated, and with excess reagent catalytic activity was almost completely abolished (44)- Similar results were obtained with FDNB (15). The reactive sulfhydryl groups may be located in apolar regions of the enzyme molecule since they were not affected by N-ethylmaleimide or iodoacetic acid. 

The activation of liver FDPase by a variety of sulfhydryl reagents has been examined by Little et al. (46), and their results generally confirm those reported by Pontremoli and his co-workers. In the disulfide exchange reaction 5,5 -dithio-bis(2-nitrobenzoic) acid was most effective (45), and in general, the disulfides were more effective than reagents such as p-mercuribenzoate or iodoacetamide. The nature of the group introduced appears to affect the conformation of the modified enzyme. 

Table 8. Quantitative aspects of the reaction of p-mercuribenzoate (PCMB) with C. pasteurianum ferredoxin...

Investigate the inhibition of the enzymatic reaction by p-mercuribenzoate at concentrations of about 10 M Show how an investigation of inhibition or poisoning of the catalyst might be used to establish the number of catalytically active sites on an invertase molecule. 

A very important conclusion was reached based on the effect of p-mer-curibenzoate on the NADPH oxidase and the NADPH-cjrtochrome c reductase activities of microsomes, namely, that the natural acceptor might be a component reactive with oxygen and involved in hydroxyla-tions or demethylations (11). It was found that in the absence of cytochrome c, the oxidase activity was largely inhibited by p-mercuri-benzoate. In the presence of cytochrome c, NADPH oxidation exceeded cytochrome c reduction in the absence of p-mercuribenzoate and the two rates equaled each other in the presence of p-mercuribenzoate. Thus, a mercurial sensitive oxidase distinct from the reductase was indicated, and this component was hypothesized to be connected with hydroxylation and/or demethyktion (11). 

The influence of mercurials on the NADPH-cytochrome c reductase activity is complex. The activity in microsomes is stimulated about 50% by p-mercuribenzoate (11). Mersalyl inhibits the NADPH-cytochrome c reductase activity (S87). 

The lipase-solubilized reductase is inhibited by p-mercuribenzoate, is protected from this inhibition by NADPH, and the inhibition is relieved by thiols (10). Careful titration of this enzyme with p-mercuribenzoate at pH 6.5 results in an almost 3-fold stimulation upon addition of 2 moles of mercurial per flavin the control activity is again observed when 7 equivalents have been added. At pH 7.7, a stimulation of 70% is seen with 1 equivalent and loss of activity is complete (extrapolated) with 6 equivalents (245). The protection of the enzyme by NADPH against mercurial inhibition is reminiscent of the effects with NADH cytochrome 63 reductase (360). 

That hydroxylamine might not be an obligatory intermediate, or occur as a free intermediate, in the reduction of nitrite to ammonia is suggested by the properties of nitrite reductases of Azotobacter chroococcum and Escherichia coli. The former is an adaptive enzyme, the formation of which requires nitrate or nitrite in the culture (31,2). It is FAD-depen-dent and presumably contains metals and p-mercuribenzoate inhibitable... 

The nitrite reductase system of Achromobacter fischeri appears to be composed of two separable enzymes (341). The first enzyme is a flavin reductase and utilizes NADH or NADPH to reduce FMN or FAD. The second interacts with the flavin reductase and converts nitrite and hy-droxylaraine to ammonia. The nitrite reductase enzyme has a molecular weight of 95,000 4,000 (Table XVII), contains two heme c per mole, and is inhibited by p-mercuribenzoate, cyanide, and carbon monoxide. 

As already oudined, inhibition is essentially complete when caused by reagents that react with sulfhydryl groups (for example, p-chloro-mercuribenzoate, p-mercuribenzoate, o-iodosobenzoate, L-ascorbic acid silver, cupric, and mercuric ions iodine, and ferricyanide) this inactivation can be reversed to some extent by hydrogen sulfide and by cysteine. Lineweaver—Burk graphs have shown that the action of L-ascorbic acid is noncompetitive, and L-ascorbic acid acting in the presence of cupric ions probably causes formation of an inactive cuprous-enzyme. The action of p-chloromercuribenzoate on barley beta-amylase has been shown to be a competitive inhibition. In contrast, the soya-bean p-chloromercuribenzoate inhibition is noncompetitive, and the extent of inhibition is inversely related to the concentration of acetate ion. The latter exhibits a protective effect, and there... 

The remainder of this chapter deals, in the main, with the preparation of a number of derivatives of the cysteinyl residue such as the acidic compounds, cysteic acid, S-carboxymethylcysteine, S-sulfo-cysteine, and the S-sulfenylthiosulfate of cysteine, the basic derivative S-aminoethylcysteine and S-carboxamidomethylcysteine. Procedures are also presented for the application of N-ethylmaleimide, p-mercuribenzoate, 5,5 -dithiobis(2-nitrobenzoate), and azobenzene-2-sulfenyl bromide to the spectrophotometric determination of sulfhydryl groups. 

Reaction of thiols with organomercurials 3.8.12.1. Titration with p-mercuribenzoate (PMB)... 

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