Alkaline phosphatase, hydrolytic enzyme

Pantothenate in blood and tissues is bound (R9) and released by autolysis or hydrolysis. More vitamin could be released by use of an alkaline phosphatase and an enzyme from avian liver (L6). This method liberates pantothenate from coenzyme A in a variety of foods and tissues (N3, N4). A comparison of hydrolytic methods in blood suggested autolysis to be the most advantageous method (N3) in our hands, treatment with Clarase gave more reliable results as compared with autolysis, acid hydrolysis, treatment with Mylase P, or combination of Clarase and papain, or liver enzyme and alkaline phosphatase. In urine, pantothenic acid is unbound our results show no increase with Clarase treatment. The vitamin has presumably a low threshold. Pantothenic acid shows the same concentration in blood and cerebrospinal fluid. 

FIGURE 2-21 The pH optima of some enzymes. Pepsin is a digestive enzyme secreted into gastric juice trypsin, a digestive enzyme that acts in the small intestine alkaline phosphatase of bone tissue, a hydrolytic enzyme thought to aid in bone mineralization. 

Although inversion was not observed with the E. colt alkaline phosphatase, it has been observed for ribonucleases and many other hydrolytic enzymes and for most kinases transferring phospho groups from ATP. The difference lies in the existence of a phospho-enzyme intermediate in the action of alkaline phosphatase (see Eq. 12-38). Each of the two phosphotransferase steps in the phosphatase action apparently occurs with inversion. The simplest interpretation of all the experimental results is that phosphotransferases usually act by in-line -like mechanisms which may involve metaphosphate-ion-like transition states that are constrained to react with an incoming nucleophile to give inversion. An adjacent attack with pseudorotation would probably retain the original configuration and is therefore excluded. 

These enzymes, which mainly catalyze hydrolytic reactions, have the zinc ions at their active sites. However, Zn ions also appear necessary in some cases for stabilization of the protein structure, e.g. in Cu/Zn SOD, insulin, liver alcohol dehydrogenase and alkaline phosphatases. 

The amino acid sequence around the serine that is phosphorylated in the presence of inorganic phosphate at low pH can be seen in Table III (55-57). The sequence of Schwartz et al. (55) accounted for 56% of the peptides that contained 32P (20% or more of the peptides were excluded as extreme fractions when the peaks were pooled). The sequence, as far as it is known, is the same for alkaline phosphatase from a mammalian source (58). It is interesting to note, as pointed out by Boyer and others (59-64), that many hydrolytic enzymes with a serine residue at their active site have the same general sequence, i.e., Asp (Glu)-Ser-Ala (Gly). 

Many of the E. coli psi genes function to enhance Pi availability in, and uptake from, the external medium. For example, phosphate starvation induces pho A whose product is alkaline phosphatase, a hydrolytic enzyme that is excreted into the periplasmic space where it acts to cleave extracellular organic P to Pi. A second psi gene system, the phosphate-specific transport (Pst) operon uses energy to transport Pi across the E. coli membrane. The affinity of this four-gene transport system is much greater than that of the constitutive Pi shuttle. Many of these same molecular starvation rescue mechanisms have been characterised in yeast. 

There are other hydrolytic enzymes, such as lipases (see below) and alkaline phosphatase, with a mechanism closely related to that of the serine proteases or glyceraldehydephosphate dehydrogenase (GAP-DH) containing a cysteine in the active site. 

Kimura and his associates have been preeminent in exploiting the potential of Zn(II) complexes of pendant-arm polyaza macrocycles to act as models for the hydrolytic Zn(II)-containing enzymes. Collectively, their work in this area involves structurally unmodified macrocycles as well as pendant-arm macrocycles, and the reader is referred to a number of reviews 6-15) that summarize their work in its entirety. The particular object of this section is to examine how different types of pendant arm have been introduced onto a macrocyclic framework and how it has been possible to utilize their presence to elicit information of relevance to a particular group of enzymes. The enzyme groups studied using pendant-arm macrocycles have been the alkaline phosphatases and the class II aldolases. 

Another important hydrolytic enzyme of the gut is acid phosphatase Like enterokinasc, it is bound to the enierocyte facing the lumen and is present in the duodenum, jejunum, and ileum. Alkaline phosphatase, a zinc metalloenzyme, also occurs in the gut. Acid phosphatase and alkaline phosphatase catalyze the removal of phosphate groups from a wide variety of compounds in foods, for example, sugar phosphates, triose phosphates, nucleotides such as AMP, ADP, and ATP, pyrophosphate, and phosphorylaled amino adds, A number of sugar and triose phosphates are described in the section on glycolysis in Chapter 4,... 

Several independent techniques have been used to identify bacterial alkaline phosphatase activity in natural bacterial assemblages. Phosphatase activity perse has often been detected using para-nitrophenyl phosphate as a substrate enzyme activity is expressed as rate of formation of the hydrolytic product, para-nitrophenol, which is easily detected spectrophotometrically because it absorbs light strongly at = 395 10 nm at pH > 9 (see Huber and Kidby, 1984, for a review of variations on this theme). Other substrates that have been used to detect alkaline phosphatase activity... 

As noted in the introduction, the effects of multiple modes of catalysis are often multiplicative rather than simply additive. Consequently, it is not surprising that a number of hydrolytic metalloenzymes have evolved that utilize a constellation of three metal ions in catalysis. Perhaps not coincidentally, all well-characterized examples of this class catalyze the hydrolytic cleavage of phosphate ester or phosphoric acid anhydride bonds, which represent a difficult and long-standing chemical problem. In every case but one, the metal ions in the trimetal centers are all zinc. As we shall see, alkaline phosphatase utilizes a Zn2Mg trinuclear center. It should be pointed out that in the older literature many of the enzymes discussed in this section have been described as containing dinuclear metal centers. Only in the last few years has it become clear that three metal ions are present and participate in catalysis by these systems. 

It is likely that alkaline phosphatase has the longest history and is the most intensively investigated hydrolytic enzyme discussed in this chapter. Because most of the published work has, however, been discussed in detail in earlier reviews, this discussion will be limited to a very brief summary of the most salient features and the latest findings. Alkaline phosphatases are zinc-dependent enzymes that are rather nonspecific and exhibit maximal catalytic activity at alkaline pH (>7.5). The enzyme is found in all organisms examined to date, and in humans the level of serum alkaline phosphatase activity is a clinically important indicator of a variety of disease states. 

Fig. 1.4 Examples of dinuclear complexes mimicking hydrolytic enzymes, a Urease [68] b GpdQ [10] c Alkaline phosphatase [69] d PAP [70] e MpL [71] f Aminopeptidase [72]...

In the reactions of hydrolytic enzymes, like chymotrypsin or alkaline phosphatase, rapid sampling and optical observations of the fate of chromophoric substrates, gave complementary information. The recording of the time course of the formation of an enzyme-product complex, as well as of free product helped to map out the reaction mechanism. 

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