Phosphate, p-nitrophenyl, hydrolysis

These were differently affected by different procedures. For example, when the enzyme was activated at 55°, the increment in ki was slight, but k2 increased 3.5-fold. Similarly, in the presence of EDTA, fc, and k2 values decreased independently, suggesting that the sites for both activities were different. Center and Behai (5) found that with the P. mirabilis enzyme, cyclic 2, 3 -UMP competitively inhibited the hydrolysis of bis(p-nitrophenyl) phosphate. The Ki was 40 pAf very close to the Km for the cyclic nucleotide (Km, 75 yM) which indicated that the two compounds could serve as alternate substrates being hydrolyzed at the same active site. In contrast, 3 -AMP was a mixed inhibitor of cyclic 2, 3 -UMP and bis(p-nitrophenyl) phosphate hydrolysis. Adenosine was a mixed inhibitor of bis(p-nitrophenyl) phosphate hydrolysis but a competitive inhibitor of 3 -AMP hydrolysis. From such kinetic studies Center and Behai (5) suggested that two separate and adjacent sites A and B are involved in the hydrolysis of the diester and phos-phomonoester substrates. Site A serves as a binding site for hydrolysis of ribonucleoside 2, 3 -cyclic phosphates and together with site B catalyzes the hydrolysis of the diester bond. During this reaction 3 -... 

Inhibition of p-Nitrophenyl Phosphate Hydrolysis by (+)-Tartaric Acid and K of p-Nitrophenyl Phosphate at Various pH Values 6... 

Deming and Pardue studied the kinetics for the hydrolysis of p-nitrophenyl phosphate by the enzyme alkaline phosphatase. The progress of the reaction was monitored by measuring the absorbance due to p-nitrophenol, which is one of the products of the reaction. A plot of the rate of the reaction (with units of pmol mL s ) versus the volume, V, (in milliliters) of a serum calibration standard containing the enzyme yielded a straight line with the following equation... 

The hydrolysis of p-nitrophenyl acetate and bis(p-nitrophenyl phosphate) are frequently used to probe hydrolytic activity. A problem with some other dinuclear systems is that the Zn units are held together by bridging ligands which can be cleaved on reaction with the substrate.440 This is not the case in a ditopic ligand such as those designed by Lippard and co-workers based on Kemp s triacid imide with a xylyl spacer.441,442 Both zinc dimers and mixed metal dimers were formed and a structure characterized with a bridging phosphodiester (Figure 6). 

In related work a library of 1,458 peptide ligands and various metal salts was tested in hydrolysis reactions of (p-nitrophenyl)phosphates.35 An active substructure composed of polymer-bound histidine in combination with Eu3+ was identified by further dissecting the original hit structure. It needs to be pointed out that catalytically active polymer beads can also be tested for catalytic activity using IR-thermography. In a seminal paper this was demonstrated using 7,000 encoded polymer beads prepared by split-and-pool methods, specifically in the metal-free acylation of alcohols.36... 

Also, Chin s dihydroxy-bridged dicobalt(III) complex 10 provides one of the largest rate enhancements ever observed for phosphodiester hydrolysis [36]. Reaction with the activated phosphodiester methyl(p-nitrophenyl)phosphate (MNPP) yields 11. A crystal structure is available for the analogous dimethylphosphate complex in which the Co ions are 2.9 A apart. At pH 7 and 45 °C 11 releases p-nitrophenolate with k = 0.1 s 1 which corresponds to a nearly 10I2-fold rate enhancement over spontaneous hydrolysis of the substrate. The product of the reaction is a methylphosphate-bridged complex. Based on the pH rate profile and on... 

In spite of the above mentioned Co(EII) compounds, kinetically labile metal complexes may provide fast product/substrate exchange and some of these systems show real catalytic activity. In native dinuclear phosphatases Mg(II), Mn(II), Fe(II/III), or Zn(II) ions are present in the active centers. Although the aqua complexes of the weakest Lewis acids, Mg(H) and Mn(II), show measurable acceleration of e.g. the transesterification of 2-hydroxypropyl p-nitrophenyl phosphate HPNP, [Mn(II)] = 0.004 M, kobs/ uncat = 73 at pH 7 and 310 K, [38] or the hydrolysis of S -uridyluridine (UpU) [39], only a few structural [40] but no functional phosphatase-mimicking dinuclear complexes have been reported with these metal ions. 

Schultz s group employed an a-hydroxyphosphonate hapten [99] and subsequently isolated 20 cell lines of which 5 catalysed the hydrolysis of the model substrate p-nitrophenyl phosphate [100] above background (Fig. 34) (Scanlan et al., 1991). Antibody 38E1 was characterized in more detail and kinetic parameters were afforded by Lineweaver-Burke analysis. This antibody exhibited 11 turnovers per binding site with no change in Vmax, and thus acted as a true catalyst. Moreover, examination of substrate specificity showed that catalysis was entirely selective for p-substituted species (Appendix entry 6.6). 

Fig. 34 (above) Antibody 38E1, generated from the a-hydroxyphosphonate hapten [99], catalysed the hydrolysis of p-nitrophenyl phosphate [100]. 

F. R. Murphy, V. Krupta, G. S. Marks, Drug-Induced Porphyrin Biosynthesis. XIII. Role of Lipophilicity in Determining Porphyrin-Inducing Activity of Aliphatic Amides after Blockade of Their Hydrolysis by Bis-(p-nitrophenyl)phosphate , Biochem. Pharmacol. 1975, 24, 883-889. 

A hydroxoaqua copper complex containing N, N, N, A -tetramethyl-1,2-diamino-ethane (250) is an excellent catalyst for the hydrolysis of sarin, O-isopropyl methylphosphonofluoridate (251), and diethyl p-nitrophenyl phosphate (252 R = Et). The mechanism of the reaction probably involves bound hydroxide attacking the phosphoryl group with concomitant electrophilic catalysis by copper. 

UMP becomes bound to site B which catalyzes the hydrolysis of the phosphomonoester bond. Adenosine and 3 -AMP by binding at site B could interfere with the breakdown of cyclic 2, 3 -UMP. Similarly, binding of bis (p-nitrophenyl) phosphate at site A could interfere with the breakdown of 3 -AMP. Cyclic 2, 3 -UMP and bis(p-nitrophenyl) phosphate compete for site A while adenosine competes with 3 -AMP for site B. Unemoto et al. (7) have examined the mutual inhibition of substrates and substrate analogs for the enzyme from halophilic V. alginolyticus. They also concluded that 3 -ribonucleotides and ribonucleo-side 2, 3 -cyclic phosphates are hydrolyzed at different sites. However, because of the nature of the mutual inhibition between 3 -AMP and bis(p-nitrophenyl) phosphate, they suggested that part of the site for the latter substrate overlaps with the 3 -nucleotidase site. At this time the precise mechanism of action of the enzyme is not settled, but clearly there are two active sites, one a 3 -nucleotidase site and a cyclic phosphate diesterase site. Anraku (18) has described this protein as a double-headed enzyme. 

By quenching enzyme solutions during the hydrolysis of 32P-ATP and 32P-AMP at high substrate concentrations (>10-3 M), Lazdunski et al. 138) found that two moles of phosphate are bound per mole of enzyme at low pH and one mole per mole of enzyme at alkaline pH. In contrast, by means of a rapid sampling apparatus, Reid and Wilson found only 0.1 mole of phosphate bound per mole of enzyme during the hydrolysis of 32P-ATP, 32P-PP1( and 32P-p-nitrophenyl phosphate at low substrate concentrations (<10"4 M) and alkaline pH 139). 

In contrast, Reid and Wilson (139) found that at low concentrations of ATP, PPi, and p-nitrophenyl phosphate (10-5 M) if the enzyme-substrate solution at pH 8.0 is rapidly quenched during steady state hydrolysis, 0.1 mole of phosphate is bound per mole of enzyme. This result would... 

For the hydrolysis of p-nitrophenyl phosphate by placental phosphatase at pH 10.5 the corresponding figure is 10,380 cal/mole (101a). Taking into account changes in ionization of the enzyme, a value of 9800 cal/mole for 4-methylumbelliferyl phosphate and calf intestinal phosphatase was derived (143). The comparable values for nonenzymic hydrolysis of monoanions of aryl phosphates are 27,000-31,000 cal/mole... 

Most of these observations have since been verified. Phosphorylation by substrate has been shown to occur under acid conditions by using a stopped-flow technique (118, 165) as illustrated in Fig. 4. Under alkaline conditions the phosphoryl enzyme cannot normally be observed or isolated because the rate of dephosphorylation exceeds the maximum rate of phosphorylation (170). One interesting aspect is that the pH-rate profiles for phosphorylation and dephosphorylation are quite different, as is the case for E. coli alkaline phosphatase (171). Barman and Gut-freund studied the formation and breakdown of milk phosphoryl phosphatase using a rapid-quenching technique and concluded that dephosphorylation could not be rate limiting for the hydrolysis of p-nitrophenyl phosphate at pH 7 (S3). 

Figure 1 (38) illustrates that maximal hydrolysis of phenyl phosphate occurred at pH 4.9, 5.0, and 5.0 in acetate, citrate, and tris-HCl buffers, respectively. For p-nitrophenyl phosphate, the corresponding values were 4.9, 4.7, and 5.5. For / -glycerophosphate, the values were 5.5, 5.7, and... 

Relative rates of hydrolysis were determined with 0.5 ml reaction mixtures in 0.1 M sodium acetate buffer, pH 5.0, at 37°. Liberated phosphate was measured by the method of C. H. Fiske and Y. SubbaRow [JBC 66, 375 (1925)]. The amounts of enzyme used were 0.22 unit of crystalline enzyme and 0.24 unit of peak II enzyme. The concentration of substrate and inhibitor was 1.0 mM. For inhibitor study, 1.0 mM p-nitrophenyl phosphate was used as substrate. Inhibition was calculated from the amount of p-nitro-phenol released and expressed as fractional inhibition. 

A Lineweaver-Burk plot of the hydrolysis of p-nitrophenyl phosphate... 

A full consideration of the mechanism of the sodium pump requires an account of the role of the lipid, the binding sites for Na+, K+, Mg2+ and ATP, the mechanism of hydrolysis of ATP and the way in which this is coupled to the transport of the cation. In addition it should be noted that the enzyme also functions as a K+-dependent phosphatase, a reaction usually studied with p-nitrophenyl phosphate as substrate. Studies with inhibitors have been informative, notably with ouabain and with vanadate. Ouabain binds at one site per pump and so has been of value in quantitatively defining the enzyme in various preparations. 

An example of an optical enzyme sensor (Arnold, 1985) in a bifurcated optical fiber is shown in Fig. 9.32. The bifurcated fiber delivers and collects light to and from the site of the enzymatic reaction. The enzyme, alkaline phosphatase (AP), catalyzes hydrolysis of p-nitrophenyl phosphate to p-nitrophenoxide ion which is being detected (A = 404 nm). 

Randolph et al. (1985) Batch Hydrolysis of disodium p-nitrophenyl phosphate to p-nitrophenol Alkaline phosphatase... 

Proteinoid microspheres containing zinc hydrolyze the natural substrate, adenosine triphosphate (ATP) as well as the unnatural substrates, p-nitrophenylacetate or p-nitrophenyl phosphate. The significance resides in the fact that the energy for most biosyntheses is provided by the hydrolysis of ATP. Zinc, magnesium and other metal salts are known to catalyze the hydrolysis of ATP 10). Proteinoid microspheres containing zinc as a cofactor have an activity for hydrolysis of ATP 11 12). 

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