Alkaline phosphatase active sites, number

It was clear for some time that a number of zinc enzymes required two or more metal ions for full activity, but in the absence of X-ray structural data the location of these metal centres with regard to one another was often uncertain. When the first 3-D structures began to appear, it became clear that the metals were in close proximity. A particular feature of many of these enzymes was the presence of a bridging ligand between two of the metal sites, usually an Asp residue of the protein, which is occasionally replaced by a water molecule. While some of the sites contain only Zn ions, several contain Zn in combination with Cu (in cytosolic superoxide dismutases) Fe (in purple acid phosphatases) or Mg (in alkaline phosphatase and the aminopeptidase of lens). 

The data relevant to the number of active sites of alkaline phosphatase can be divided into two groups One group derived from studies at low substrate concentrations (S < 10-4 M) indicates one active site per dimer, and the other group derived from studies at high substrate concentrations (S > 10 3 M) indicate two sites. 

We have already seen a number of models for the zinc(II) containing enzymes such as carbonic anhydrase in Section 11.3.2. Zinc is an essential component in biochemistry, and forms part of the active site of more then 100 enzymes, of which hydrolases (such as alkaline phosphatase and carboxypeptidase A), transferases (e.g. DNA and RNA polymerase), oxidoreductases (e.g. alcohol dehydrogenase and superoxide dismutase) and lysases (carbonic anhydrase) are the most common. In addition, the non-enzyme zinc finger proteins have an important regulatory function. In many of these systems, the non-redox-active Zn2+ ion is present as a Fewis acidic centre at which substrates are coordinated, polarised and hence activated. Other roles of zinc include acting as a template and playing a structural or regulatory role. 

Besides GS (both type I and type II), a number of other metal-dependent enzymes including, for example alkaline phosphatases, endonucleases, DNA/RNA polymerases, and so on, require two metal cations bound in the active center for their catalytic activity [43]. It has, however, to be noted that while, for instance, in polymerases and nucleases the two cations are coordinated jointly by both a conserved Asp residue and the scissile phosphate, in GS the two cations are bound by a few separate amino acid residues (see Fig. 17.5b). Moreover, the GS substrates bind to different cations (glutamate to nl and ATP to n2 NH4" " has its separate binding site close to nl). This is in line with a relatively large distance between the two cations in GS (0.6 nm), as compared to those in, for example, RNases (0.4-0.3 nm) [43]. 

A number of other enzymes which catalyze the hydrolysis of phosphoesters are of biological importance. These include cyclic purine phosphodiesterase (little is known about its active site chemistry at present, but more shall be said about its biological role shortly) and the phosphatases. Acid and alkaline phosphatase catalyze the hydrolysis of phosphomonoesters to the corresponding alcohol and inorganic phosphate. Their pH optimums are 5.0 and 8.0, respectively hence their names. Both form covalent enzyme-substrate intermediates ... 

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