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The Structure and Mechanism of Phosphoprotein Phosphatases Many Phosphatases Require Two Metal Ions for Catalysis

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The Structure and Mechanism of Phosphoprotein Phosphatases Many Phosphatases Require Two Metal Ions for Catalysis WILLIAM P TAYLOR AND THEODORE S WIDLANSKI MINIREVIEW Charged with meaning: the structure and mechanism of phosphoprotein phosphatases Many phosphatases require two metal ions for catalysis. New structural information on two serinekhreonine phosphatases offers insight into how the metals contribute to catalysis. A comparison with the structures of protein tyrosine phosphatases, which do not use metal ions, shows that the only similarity at the active site is that of charge. Chemistry & Biology November 1995, 2:713-718 Many biological processes are regulated by J simple substrates; and (3) small-molecule specific - enzymes chcniical event - the cleavage or formation of phos- that hydrolyze one (or a group of structurally similar) phdte esters. In nature, these reactions are catalyzed by substrate(s), for example, phosphoinocitol nlonophos- tv,m sets of enzynws, phosphatases and kinnses. Kinases phatasc (this cnzyrne may be the clinical target of operate in the synthetic direction (phocphorylation) lithium ion therapy for depression [A]). while phosphatases catalyze the hydrolysis (cleavage) reaction. Over the last decade there has been a growing A second useful classification scheme is our according rcalizCition that phosphataws are extremely important in to the enzyme’s mechanism of action. Some phos- ccllulx and organismal functions [ 1 1. Here, we focus on phatascs use an active site nucleophile 3s the initi,J recent Ed\-antes in our understanding of phosphatases. phosphoryl group acceptor; others transfer it inuned- especially phosphoprotein phosphatases, with J vielv to atcly to water (Fig. 3). The first group cm be further clarifyiiig some of the mrchanictic and structural con- subdivided according to the phosphoryl group acceptor plcsitie and mlbiguities presented by this diverse group (cysteine. histidinc, or serine). Mculbers of the second of mzynies. The newly reported structural information group characteristically use J two-metal-ion dyad to on txvo wrine/threonine (Ser/Thr) phosphatases [2,3] is bind phosphate esters and catalyze their subsequent particularly enlightening. hydrolysis. Indeed, this metal ion motif is ubiquitous in phosphoryl group transfer biochemistry, as it is also ust‘d Categories of phosphatases by phosphodiesterases (e.g. nucleases [5]) and phosphc>- The substrates for phosphatases range from small phos- triesterases [ 01. Two-metal-ion phosphatxes are vt’r) phoryl,ltcd nletabolites such as glucose-(,-pllosphatc and heterogeneous, however, vxying in metal-ion type, second nlessengers (e.g. phosphoinositols) all the way up protein wqueuce, structure, ligands, active-site catalytic to large phosphorylated proteins. Over a hundred residues md cvw mechanism. For example, alkaline phosphatClses xe known, and it is likely that the total phosphatasc transfers the phosphoryl group to a serinc number of thcsc enzymes is well over a thousand, or residue, not to wClter, and has a third active-site metal (a > I ‘%I of the proteins encoded by the human genome. To Mg(II) ion that does not directly contact the phosphate m,tkc scnsc of this lxge family of enzymes, a clnssifica- ester) [7]. Zn(I1) ions are frequently found in the active tion schemr is essential (Fig. l).There are three major site of tmx-metal-ion phosph:ltases, but mmy other d- groups of phosphntases: (1) nonspecific - these valent metal ions x-e also common. Manmlalian purple enzymes mill catalyze the hydrolysis of almost any pho- acid phosphatase, which contains an unusual hinuclear ph,lte ester: (3) phosphoprotein specific - enzymes that Fe(II)-Fc(ll1) mrt<J dyad 181, sc‘rvcs ds a reminder th,it USC phosphaprotcilir or phosphopeptides as preferred the tlvo-metal-ion enzynit5 have c’nornious diversity. Fig. 1. A hierarchy of phosphatases. Initial classification is based on sub- strate specificity; secondary classifica- tion is based on mechanism. The low MW acid phosphatases and the low MW dual specificity phosphatases seem to be a related family of enzymes. 0 Current Biology Ltd ISSN 1074-5521 713 714 Chemistry & Biology 1995, Vol 2 No 11 Fig. 2. Typical phosphatase reaction (a) WJ ROH mechanisms. (a) Direct transfer to \t water, normally catalyzed by active- E + RO-PO,‘- +==- E.RO-PO,‘- - E*Pi yE+Pi site metal ions. (b) Hydrolysis via a phosphoenzyme intermediate. Serine, cysteine, and histidine are nucleo- philes commonly used as the initial phosphoryl group acceptor. (b) ROH E + RO-PO, 2---- EOR@pO zmL E-PO 2_“L EmPi A E + Pi 3- 3- - Phosphoprotein phosphatases an enzyme in the class and the name of the class) specifi- Both phosphoprotein phosphatases and protein kinases cally dephosphorylate the B subunit of phosphorylase are important in the regulation of the phosphorylation kinase and are inhibited by two low molecular weight state of proteins. Often, phosphatases simply reverse the proteins, inhibitor-l (I-l) and inhibitor-2 (I-2). Type 2 effects of kinases, for instance by inactivating a protein enzymes prefer the (X subunit of phosphorylase kinase as a that has been activated by phosphorylation. But things substrate and are insensitive to I-l and I-2. The type 2 are not always so straightforward. Phosphatases can also enzymes are subdivided according to their divalent metal regulate kinase activity, thus indirectly regulating the ion requirements. PP-2A enzymes (like PP- 1 enzymes) phosphorylation state of the substrates of the kinase. are stimulated by Mn(II), whereas PP-2B enzymes and Some phosphatases contain SH2 domains which specifi- PP-2C enzymes use Ca(I1) and Mg(II), respectively. N-1, cally recognize and bind to phosphotyrosine residues, PP-2A, and PP-2B enzymes have highly homologous and their activity is therefore modulated by protein catalytic domains, and the differences between their activ- tyrosine kinases. Such examples of crosstalk allow for the ities are mostly caused by regulatory subunits bound to exquisite fine tuning of signal transduction cascades the catalytic domain. PP-2C enzymes, however, appear to necessary for regulating cell function. be monomeric, and are structurally unrelated to the other Ser/Thr phosphatases. Phosphoprotein phosphatases are subdivided into the Ser/Thr phosphatases 191, which are probably all two- Much is known about the biological roles of some of the metal-ion phosphatases, the protein tyrosine phosphatases Ser/Thr phosphatases, particularly those of the type 1 (PTPases) [ 101, and the dual-specificity phosphoprotein family. Early studies on the regulation of glycogen syn- phosphatases [l l-131. PTPases and dual-specificity thesis and breakdown provided seminal information on phosphoprotein phosphatases are mechanistically related the effect of protein phosphorylation/dephosphorylation and use an active-site cysteine located in a phosphate on the activities of individual enzymes involved in meta- binding loop as the phosphoryl group acceptor. True bolic and catabolic processes [ 171. Dephosphorylation by DTPases are highly specific for phosphotyrosine, whereas PP-1 reverses the activity of protein kinase A and regu- the dual-specificity enzymes hydrolyze both phospho- lates the phosphorylation state (and therefore the activ- tyrosine and phosphoserine (the low-molecular-weight ity) of three of the key enzymes involved in glycogen dual-specificity phosphatases prefer aromatic phosphate metabolism: phosphorylase kinase, glycogen synthase and esters and phosphotyrosine substrates, but can still often glycogen phosphorylase. The activity of PI’-1 is itself hydrolyze phosphoserine-containing peptides and pro- tightly regulated by levels of cyclic AMP, and the degree teins [14]). For most phosphatases the nature of the of phosphorylation of the inhibitors I-l and I-2. These physiologically rel evant substrate(s) is unknown. Thus, and other inhibitors of PP-1 (such as the polyether, the fact that a phosphatase is classified as having Ser/Thr okadaic acid, and the cyclic heptapeptide toxins known phosphatase activity (Fig. 1) does not necessarily imply as microcystins) have been useful for the study and that this is its sole activity itz viva. Indeed, calcineurin, a manipulation of signal transduction cascades. Studies prototypical Ser/Thr phosphatase, can hydrolyze phos- on inhibitors of calcineurin-A (PP-2B), such as the photyrosine-containing peptides as well as aromatic cyclosporins and FK506, have added greatly to our phosphates such as y-nitrophenyl phosphate [15]. Even understanding of T-cell activation and the design of new prostatic acid phosphatase, a so-called nonspecific phos- immunosuppressants [ 1X]. phatase, has phosphoprotein substrates (in particular, those containing phosphotyrosine) with K, values in the Structure and mechanism low nanomolar range ]I 61; perhaps a phosphoprotein is a Despite a wealth of information about the biological major target in vim. processes involving Ser/Thr phosphatases, chemical and structural information about these enzymes is sparse and Functions of Ser/Thr phosphoprotein phosphatases little is known about how they catalyze phosphate ester The Ser/Thr phosphatases fall into four classes, which hydrolysis. Most of the mechanistic information on these account for virtually all cellular Ser/Thr phosphatase proteins stems from mutagenesis studies of bacteriophage activity [9].Type 1 enzymes (PP-I enzymes; PP-1 is both X phosphatase, a phosphatase with substantial homology Phosphoprotein phosphatases Taylor and Wdlanski Fig. 3. Actiw-site structure of two phosf,hol)rot’iri l)hosl)h,lt,l~c,~. (a) Kiblwn cliagram of PI’-1 sho\z/ing lhcl ~-u-~-u-~
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