Alkaline phosphatase histidine

Brydon and Roberts- added hemolyzed blood to unhemolyzed plasma, analyzed the specimens for a variety of constituents and then compared the values with those in the unhemolyzed plasma (B28). The following procedures were considered unaffected by hemolysis (up to 1 g/100 ml hemoglobin) urea (diacetyl monoxime) carbon dioxide content (phe-nolphthalein complex) iron binding capacity cholesterol (ferric chloride) creatinine (alkaline picrate) uric acid (phosphotungstate reduction) alkaline phosphatase (4-nitrophenyl phosphate) 5 -nucleotidase (adenosine monophosphate-nickel) and tartrate-labile acid phosphatase (phenyl phosphate). In Table 2 are shown those assays where increases were observed. The hemolysis used in these studies was equivalent to that produced by the breakdown of about 15 X 10 erythrocytes. In the bromocresol green albumin method it has been reported that for every 100 mg of hemoglobin/100 ml serum, the apparent albumin concentration is increased by 100 mg/100 ml (D12). Hemolysis releases some amino acids, such as histidine, into the plasma (Alb). 

In enzymes, the most common nucleophilic groups that are functional in catalysis are the serine hydroxyl—which occurs in the serine proteases, cholinesterases, esterases, lipases, and alkaline phosphatases—and the cysteine thiol—which occurs in the thiol proteases (papain, ficin, and bromelain), in glyceraldehyde 3-phosphate dehydrogenase, etc. The imidazole of histidine usually functions as an acid-base catalyst and enhances the nucleophilicity of hydroxyl and thiol groups, but it sometimes acts as a nucleophile with the phos-phoryl group in phosphate transfer (Table 2.5). 

Photooxidation of alkaline phosphatase in the presence of methylene blue and Rose Bengal causes loss of activity for both native and apo-enzyme. In the case of the native enzyme, zinc protects 2 to 3 of the 16 histidine residues. The rate of oxidation of tryptophan is not affected by zinc, and there was no loss of tyrosine. Also, photooxidation of the apoenzyme diminishes zinc binding. It would appear that histidine residues play a role in binding the two zinc ions necessary for enzymic activity (91). 

Treatment of the enzyme with A-bromosuccinimide oxidized 2 of the 8 tryptophan residues and 8 of the 20 tyrosine residues, but none of the histidine residues. This treatment causes the phosphotransferase activity with tris as an acceptor to double and the hydrolase activity to increase slightly. In the case of cobalt alkaline phosphatase, the above treatment caused a threefold increase in hydrolase activity and the generation of an even greater phosphotransferase activity (91). 

The serine group which becomes phosphorylated does not appear to possess any marked nucleophilic reactivity, nor is there any evidence that a histidine group participates as a general acid-general base catalyst. Rate constants for the nonenzymic hydrolysis of alkyl and aryl phosphate monoanions at 25° are in the range HP7 to 10-9 sec-1 (167), while the comparable alkaline phosphatase-catalyzed values (in this case they refer to dianions) are in the range 102 to 103 sec-1. Thus one has to account for a rate enhancement factor of 109 to 1012. Moreover, the... 

The active sites of these enzymes can have a nitrogen ligand, usually as histidine (acid phosphatases and some protein phosphatases), a nucleophilic serine residue (alkaline phosphatases), a cysteine residue in which the thiol group can form a covalent species with the phosphate ester (protein phosphatases), or an aspartate-linked phosphate (plasma membrane ion pumps). The inhibitory form of vanadium is usually anionic vanadate V(V), but cationic vanadyl V(IV) has also shown strong inhibition of some types of phosphorylase reactions. Above neutral pH, speciation of vanadyl ions produces anionic V(IV) species capable of inhibition of enzymes in the traditional transition-state analogue manner [5],... 

Several proteins and enzymes have innate affinity in their native state for the metal chelate supports due to the presence of one or more surface histidines (Table 3). Coulet et al. [154] immobilized lactate dehydrogenase, malate dehydrogenase and alkaline phosphatase on Co2+, Zn2+, Cu2+-chelate Sepharose. All the enzymes were active in the immobilized state although the retention of the specific activity was quite low. Among the enzymes used, alkaline phosphatase retained higher activity and among the various metal chelates maximum activi-... 

Amino acids have been studied in relation to alkaline phosphatase, and organ differences have been observed by Bodansky (B30) and Fishman (F13). Inhibition is competitive in nature. The most recent study on the influence of L-histidine demonstrates alteration in the pH optimum (B33) of hydrolysis. Moreover, a survey of amino acid inhibitors (Fll) has produced (F13) the unique stereospecific uncompetitive inhibitor, l-phenylalanine, which is discussed in detail in section 3.1.6. 

The chemical shift of the phosphorus resonance of various nucleotides has been studied as a function of pH in the presence and absence of RNase A. The signal shifts upheld on protonation of the phosphate and the apparent pATa of the phosphate group in 2 -CMP complex with RNase is the same as the pATa of histidine-119 in this enzyme as determined by n.m.r. From n.m.r. relaxation rates for the ternary complex manganese(n)-phosphate-E. coli alkaline phosphatase, it has been concluded that an outer-sphere complex is formed which has a shorter lifetime than the enzyme turnover rate. The latter conclusion is consistent with the participation of the complex in the enzymic reaction. 

Zinc may also have a regulatory role i.e., it does not participate in the various catalytic steps, but its presence increases the catalytic rate. This is a rather loose but common definition. Typically, zinc in the B site of alkaline phosphatase (Section V.B) has such a role, and the ligands are histidines, aspartates, and water molecules. 

It is noteworthy that three His, Glu, Asp or Cys residues provide zinc ligands for all known enzyme catalytic zinc sites [ 30], Water is the fourth ligand and histidine is by far the most frequent amino acid among the catalytic site residues. Three histidines are found in human carbonic anhydrases 1 and II, p-lactamase, the DD-carboxypeptidase of Streptomyces albus G, adenosine deaminase and astacin [30]. Two histidines are characteristic of bovine carboxypeptidases A and B, thermolysin and Escherichia coli alkaline phosphatase... 

Enzymes may use any of the above mentioned modes of catalysis in order to catalyze a particular chemical reaction. For example, the imidazole ring of a histidine residue of the enzyme a-chymotrypsin (Section 4.4) can function as a general-base catalyst, while in the enzyme alkaline phosphatase, the same residue can function as a nucleophilic catalyst. Indeed, enzymes are complex catalysts which employ more than one catalytic parameter during the course of their action. It is by this successful integration of a combination of individual catalytic processes that a rate enhancement as high as 10 " may be achieved. Furthermore, it is this combination of factors which results in a specific catalyst. 

Escherichia coli alkaline phosphatase, a dimeric zinc metalloenzyme ( 95,500 Da), binds 2 Zn ions and 1 Mg ion per monomer and functions in the non-specific hydrolysis of phosphate monoester. NMR (mi labeled histidine biosyntheti-cally incorporated into AP in conjunction with substrate NMR and Cd NMR methods were used for the assigmnent of the three Cd resonances to specific sites per monomer and their role in substrate binding. A full account of these studies can be found in the following references [6,25,80,82,201],... 

NMR spectrum of the [y- C]-histidine labeled Cd diphosphoryl alkaline phosphatase. Cd AP-P2 with spin I = 0... 

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