Alkaline phosphatase active-site structure

In general there are three phosphatase families alkaline, acid, and protein phosphatases. Alkaline phosphatases are typically dimers that contain three metal ions per subunit and have a pH optimum pH above 8. Acid phosphatases exhibit an optimum pH<7 and are usually divided into three classes low molecular weight acid phosphatases (<20 kDa), high molecular weight acid phosphatases (50-60 kDa), and purple acid phosphatases (which contain an Fe-Fe or Fe-Zn center at the active site). Phosphatases specific for I-l-P appear to be most similar (in kinetic characteristics but not in mechanism) to the alkaline phosphatases, but their structures define a superfamily that also includes inositol polyphosphate 1-phosphatase, fructose 1, 6-bisphosphatase, and Hal2. The members of this superfamily share a common structural core of 5 a-helices and 11 (3-strands. Many are Li+-sensitive (York et al., 1995), and more recent structures of archaeal IMPase proteins suggest the Li+ -sensitivity is related to the disposition of a flexible loop near the active site (Stieglitz et al., 2002). 

Mammalian alkaline phosphatase catalysis requires active site structure stabilization via the N-terminal amino acid microenvironment. Biochemistry 45 9756-9766... 

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). 

Currently, only a handful of examples of unique protein carboxylate-zinc interactions are available in the Brookhaven Protein Data Bank. Each of these entries, however, displays syn coordination stereochemistry, and two are bidentate (Christianson and Alexander, 1989) (Fig. 5). Other protein structures have been reported with iyw-oriented car-boxylate-zinc interactions, but full coordinate sets are not yet available [e.g., DNA polymerase (Ollis etal., 1985) and alkaline phosphatase (Kim and Wyckoff, 1989)]. A survey of all protein-metal ion interactions reveals that jyw-carboxylate—metal ion stereochemistry is preferred (Chakrabarti, 1990a). It is been suggested that potent zinc enzyme inhibition arises from syn-oriented interactions between inhibitor carboxylates and active-site zinc ions (Christianson and Lipscomb, 1988a see also Monzingo and Matthews, 1984), and the structures of such interactions may sample the reaction coordinate for enzymatic catalysis in certain systems (Christianson and Lipscomb, 1987). 

A detailed analysis of solid-state structures of several unrelated systems that catalyze the hydrolysis of diphosphate esters (alkaline phosphatase and the Klenow fragment , among others) revealed that their active sites invariably contain conserved carboxylate... 

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 most important aspect of the study of Co(II) metalloenzymes is the possibility of using the metal ion as a functional, built-in reporter of the dynamics of the active site. The spectral and magnetic properties of Co (II) carbonic anhydrase have given valuable clues to the catalytic function of this enzyme. The recent studies of Co(II) alkaline phosphatase and Co (II) carboxypeptidase A indicate the general applicability of this approach to enzymes where the probe properties of the constitutive metal ion are poor. The comparison of the absorption spectra of these enzymes and low-molecular weight models have shown that the proteins provide irregular, and in some cases nearly tetrahedral environments. It is obvious, however, that a knowledge of the crystal structures of the enzymes is necessary before the full potential of this method can be exploited. 

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. 

VI-IX are structurally characterised, vanadate-inhibited phosphatases. VI, Rat prostat acid phosphatase VII, bovine phosphotyrosyl phosphatase VIII, mammalian protein tyrosine phosphatase PTP-IB (mutant Cys215Ser) IX, E. coli alkaline phosphatase. For comparison, the active centre of vanadate-dependent haloperoxidases (VHPO) (V), is also shown. The structures Xa and Xb have been proposed, based on EPR, for the vanadyl complexes formed with the PTP-IB active site peptide Val-His-Cys-Ser-Ala-Gly. 

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