Nitrocatechol sulfate

Klotz and associates120 found that PEI-D-Im-type models catalyzed hydrolyses of 4-nitrocatechol sulfate by a two step mechanism resembling that of a natural enzyme. The polymer was reported to accelerate the rate by a factor of 1012 arm-pared to imidazole itself, and of 102 compared to a type-11A arylsulfatase enzyme. 

In chymotrypsin and other serine proteases the imidazole moiety of histidine acts as a general base not as a nucleophile as is probably the case in the catalysis of activated phenyl ester hydrolysis by (26). With this idea in mind, Kiefer et al. 40) studied the hydrolysis of 4-nitrocatechol sulfate in the presence of (26) since aryl sulfatase, the corresponding enzyme, has imidazole at the active center. Dramatic results were obtained. The substrate, nitrocatechol sulfate, is very stable in water at room temperature. Even the presence of 2M imidazole does not produce detectable hydrolysis. In contrast (26) cleaves the substrate at 20°C. Michaelis-Menten kinetics were obtained the second-order rate constant for catalysis by (26) is 10 times... 

The polyethylenimines are also effective in the cleavage of nitrophenyl-sulfate esters and nitrophenylphosphate esters. These have not yet been studied as extensively as the acyl esters, but interesting kinetic accelerations are already apparent. Nitrocatechol sulfate, for example, is very stable in aqueous solution at ambient temperature. In fact, even in the presence of 2 M imidazole no hydrolysis can be detected at room temperature. At 95°C in the presence of 2 M imidazole cleavage is barely perceptible. In contrast, a modified polyethylenimine with attached imidazole groups cleaves the sulfate ester at 20°C.34 Some kinetic parameters are compared in Table VI. It is obvious that accelerations of many orders of magnitude are effected by the polymer. 

TABLE VI. Comparison of Rates of Hydrolysis of 4-Nitrocatechol Sulfate... 

Substrate buffer 155.7 mg 4-nitrocatechol sulfate dipotassium salt (50 mM, Sigma) is dissolved in 10 ml sodium acetate buffer. The substrate buffer is stored at -20°C and protected from light. 

Arylsulfatase cleaves sulfate esters. Cerebroside sulfate is its natural substrate. The enzyme is also active toward p-nitrocatechol sulfate, which is the basis for this assay. 

The reaction mixture was prepared by mixing 800 fiL of substrate solution containing 0.01 M p-nitrocatechol sulfate (dipotassium salt), 1.71 M NaCl, and 0.5 M acetate buffer (pH 5.0) with 200 fiL of either leukocyte and platelet lysate or saliva. After incubation for an hour at 37°C, a 50 fiL aliquot was mixed with 100 fiL of bovine serum albumin (13 mg/mL) and 1800 fiL of cold 95% ethanol. After centrifugation to remove proteins, 20 /iL was injected into the HPLC system. The reaction was linear with up to 200 nL of lysate. 

The kinetics of arylsulfohydrolase A are quite complex, because the reaction velocity shows an abnormal relationship with the enzyme concentration and time of incubation (Roy, 1953 Baum and Dodgson, 1958) (Fig. 2), The anomalous reaction kinetics of arylsulfohydrolase A are manifested as a time-dependent decrease in hydrolytic rate during incubation with p-nitrocatechol sulfate, followed by a partial recovery of the initial rate. Baum and Dodgson (1958) have proposed that during the interaction of enzyme with substrate a new site capable of binding with substrate as well as the reaction product (nitrocatechol and sulfate) and certain other inhibitory compounds (phosphate... 

The kinetic parameters of arylsulfohydrolase A with p-nitrocatechol sulfate are shown in Table 2. Optimal activity is at pH 5.0 with a value of 0.4 mM. This enzyme is strongly inhibited by sulfate, sulfite, and phosphate ions with Kj vines of 0.052, 0.026, and 0.034 mM, respectively (Table 3). Thus sulfite is the most potent inhibitor, followed by phosphate and sulfate. Such a powerful inhibition by sulfite suggests that this ion may be a transition-state analog intermediate of the arylsulfohydrolase A catalyzed reaction. This enzyme is also strongly inhibited by ascorbic acid 2-phosphate (Carlson et ah, 1976). The inhibition by ascorbic acid 2-phosphate is competitive with a Kj value of 0.3 xM. This metabolite binds very tightly to the enzyme molecule and may serve as controlling metabolite for arylsulfohydrolases. 

As with p-nitrocatechol sulfate, arylsulfohydrolase A becomes inactivated (forms a modified enzyme) when acting on sulfolipids and ascorbic acid 2-sulfate (Roy, 1976), but no reactivation by sulfate or phosphate has been reported. It remains to be seen whether this substrate-induced inactivation has any physiological significance in sulfate metabolism. 

The kinetic properties of these multiple forms are quite similar (Farooqui, 1976b). With p-nitrocatechol sulfate as substrate, arylsulfohydrolase B has a value at least three times higher than that of arylsulfohydrolase A. Sulfate ions produce a noncompetitive inhibition of this enzyme with a K value of... 

Scheme 4 A substrate binding and WPD loop movement model for YopH with rate constants at 35 °C obtained from temperature-jump experiments. L is the ligand p-nitrocatechol sulfate. ...

Accordir to Klotz and co-workers this polymer possesses imbelievably great catalytic activity in the hydrolyris of 4-nitrocatechol sulfate 8 (122). The release of 4-nitrocatedi(d in the presence of the modified polyethyleneimine obeys typical... 

Determined with 4-methylumbelliferyl derivatives Determined with p-nitrophenyl derivatives Determined with p-nitrocatechol sulfate... 

Dodgson, K. S. Spencer, B. Impure nature of nitrocatechol sulfate. Biochim. Biophys. Acta 1956, 21, 175. 

Fig. 5. An example of Michaelis-Menten type kinetics in catalytically active synthetic polymers, from Kiefer etaL [62]. It represents the hydrolysis of nitrocatechol sulfate (XXI) by a polyethylenimmine (PEIg ),I in 0.02Af tris-buffer, pH 9.2, at 20 C. Curve 1, polymer concentration equivalent to 30 /uM imidazole groups curve 2, polymer concentration twice that for 1 curve 3, polymer concentration three times that for 1. Initial substrate concentration was 10 mM. (With permission of the Authors.)...

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