MINIREVIEW This paper is available online at www.jbc.org THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 3, pp. 1305–1308, January 20, 2006 © 2006 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. (typically five to seven), and a B value that is comparable with that of neighbor A Structural Perspective on atoms. Additional criteria involve proper O–Mϩ distances, on the average 2.4 Å for Naϩ–O and 2.8 Å for Kϩ–O pairs (4), and valence values close to unity Enzymes Activated by (5). Anomalous scattering often provides unequivocal evidence of Kϩ. * Table 1 presents a classification of Mϩ-activated enzymes based on the Monovalent Cations mechanism of activation identified from structural data and on the Mϩ Published, JBC Papers in Press, November 2, 2005, DOI 10.1074/jbc.R500023200 ϩ 1 requirement established from kinetic studies. In Type I enzymes the M func- Enrico Di Cera tions as a cofactor and the requirement is absolute. In Type II enzymes the Mϩ From the Department of Biochemistry and Molecular Biophysics, functions as an allosteric effector and the requirement is not absolute. Washington University School of Medicine, St. Louis, Missouri 63110 K؉-activated Type I Enzymes Enzymes activated by monovalent cations are abundantly represented Diol and glycerol dehydratases provide the simplest example of Type I ϩ ”in plants and the animal world. They have evolved to exploit Na؉ and K؉, enzymes. K is coordinated by five ligands from the protein and acts as a “bait readily available in biological environments, as major driving forces for for the two hydroxyl oxygens from substrate (6, 7). Enzymes involved in phos- substrate binding and catalysis. Recent progress in the structural biology phoryl transfer reactions were long recognized to be the dominant group ϩ of such enzymes has answered long standing questions about the molecu- among M -activated enzymes (2, 3), and this early observation is confirmed by ϩ lar mechanism of activation and the origin of monovalent cation selectiv- the entries in Table 1. In addition to K , these enzymes have an absolute 2ϩ ity. That enables a simple classification of these functionally diverse requirement for a divalent cation, typically Mg (1, 8–11). The mechanism of ϩ 2ϩ enzymes and reveals unanticipated connections with ion transporters. activation involves K and Mg acting in tandem to provide optimal docking for the phosphate moiety of substrate into the protein active site to enable nucleophilic attack on the P␥ or transfer of phosphate groups. The most revealing example of this strategy comes from the ATP-driven folding machine Over 60 years ago, Boyer et al. (1) reported in the pages of this Journal that ϩ ϭ ␮ GroEL, which has the highest K affinity (Kd 80 M) ever reported for a pyruvate kinase would express appreciable catalytic activity only in the pres- ϩ ϩ M -activated enzyme (9). The crystal structure of GroEL bound to ATP ence of K . A similar effect was soon discovered in other systems, and just a 2ϩ ϩ ϩ reveals Mg and K acting in tandem to assist binding of ATP to the protein few decades later the field of enzymes requiring a monovalent cation (M ) for (Fig. 1). Kϩ is coordinated by one P␣ oxygen, the backbone oxygens of Thr-30 optimal activity encompassed hundreds of examples from plants and the ani- and Lys-51, and four water molecules (12). Nucleophilic attack on the P␥ of mal world (2, 3). Since the beginning, this rapidly expanding field had to ϩ ϩ ATP is mediated by Asp-52. K fixes both groups in place through some of its address two basic questions, namely the molecular mechanism of M activa- 2ϩ ϩ ligating water molecules and is assisted by Mg , which anchors all three P tion and the structural basis of M selectivity. Because kinetic investigation groups of ATP. Branched chain ␣-ketoacid dehydrogenase kinase (13) and would only provide indirect answers, further progress in the field had to await ϩ 2ϩ ϩ pyruvate dehydrogenase kinase (14) use a similar K -Mg tandem, whereas high resolution crystal structures of M -activated enzymes, which have the molecular chaperone Hsc70 (15) and the Rad51 recombinase homolog become available only over the last decade. ϩ 2ϩ ϩ from Methanococcus voltae (16) utilize two K in tandem with Mg . Other A classification of M -activated enzymes can be based on the selectivity of variations are the Kϩ-Zn2ϩ tandem in pyridoxal kinase (17) and Kϩ coupled the effect, as established by kinetic studies, and the mechanism of activation, as 2ϩ ϩ with two Mg in fructose-1,6-bisphosphatase (18), S-adenosylmethionine shown from structural analysis. The effect has exquisite specificity, with Na ϩ ϩ ϩ synthase (19), and pyruvate kinase (20). or K being the preferred M . In general, enzymes requiring K such as ϩ ϩ ؉ kinases and molecular chaperones are also activated by NH4 and Rb but are K -activated Type II Enzymes ϩ ϩ not activated as well or at all by the larger cation Cs or the smaller cations Na ϩ ϩ ϩ Some kinases belong to Type II because K does not contact ATP directly. and Li . Enzymes requiring Na such as ␤-galactosidase and clotting pro- ϩ In this case, K exerts its influence indirectly by perturbing the conformation teases are not activated as well by Liϩ or the larger cations Kϩ,Rbϩ, and Csϩ. ϩ ϩ ϩ of active site residues. Ribokinase (21) and aminoimidazole riboside kinase (22) Because the concentration of Na and K is tightly controlled in vivo,M sdo ϩ ϩ ϩ break the K -Mg2 tandem by embracing the M in a ␤-turn adjacent to the not function as regulators of enzyme activity. Rather, they provide a driving ϩ active site. In MutL the K coordination resembles that of branched chain force for substrate binding and catalysis by lowering energy barriers in the ␣-ketoacid dehydrogenase kinase and pyruvate dehydrogenase kinase except ground and/or transition states. Enzymes activated by Mϩs evolved to take for the fact that the contact with the P␤ oxygen of ATP is replaced by a water advantage of the large availability of Naϩ outside the cell and Kϩ inside the cell molecule (23). MutL represents an intriguing intermediate between typical to optimize their catalytic function. Indeed, a strong correlation exists between ϩ ϩ ϩ Type I kinases and ribokinase that evolved a complete separation of K from the preference for K or Na and the intracellular or extracellular localization ϩ ϩ ATP. Here, one can almost envision a transition between enzymes utilizing K of such enzymes. The mechanism of M activation can be established from ϩ ϩ as a cofactor into enzymes utilizing K as an allosteric effector. crystal structures as cofactor-like or allosteric. In the former case, the M ϩ Two K binding sites have been identified in branched chain ␣-ketoacid dehy- anchors the substrate to the active site of the enzyme, often acting in tandem drogenase, one controlling the binding of thiamine diphosphate and the other, also with a divalent cation like Mg2ϩ. In such a mechanism of activation, the Mϩ is ϩ found in pyruvate dehydrogenase (24), stabilizing the quaternary structure (25). absolutely required for catalysis. In the latter, the M enhances enzyme activ- ϩ Dialkylglycine decarboxylase is a beautiful example of M -dependent molecular ity through conformational transitions triggered upon binding to a site where ϩ switch. In this pyridoxal phosphate (PLP)2-dependent enzyme K binds in a loop the Mϩ makes no direct contact with substrate. In this case, the Mϩ is not near the active site (26, 27). Substrate binding requires Tyr-301 to be in the con- absolutely required for catalysis. ϩ ϩ ϩ formation seen in the K -bound form (Fig. 1). When K is replaced by Na , the Crystallographic assignment of Naϩ or Kϩ is non-trivial, even for high res- activity of the enzyme drops 95% and the change in coordination within the loop olution structures. Naϩ has a small ionic radius (0.97 Å) and the same number causes the O␥ of Ser-80 to swing away and clash with the phenyl ring of Tyr-301 of electrons as a water molecule. Kϩ has a higher electron density but an ionic that reorients and compromises substrate binding. Likewise, in other PLP-de- radius (1.33 Å) almost identical to that of a water molecule. Hence, correct ϩ ϩ pendent enzymes like Ser hydratase (28), tryptophanase (29), and tyrosinase (30), assignment of Na and K must be based on several criteria: the presence of a ϩ the K binding site makes no contact with substrate but helps organize the archi- spherical electron density peak at a ␴ level in the F Ϫ F map considerably o c tecture of the active site. above that of all other water molecules, the number of surrounding oxygens Na؉-activated Type I Enzymes The strategy used by Kϩ-activated Type I kinases to anchor substrate to the * This minireview will be reprinted in the 2006 Minireview Compendium, which will be available in January, 2007. This work was supported in part by National Institutes of active site is also exploited by fructose-1,6-biphosphate aldolase (31) and tag- Health Research Grants HL49413, HL58141, and HL73813. 1 To whom correspondence should be addressed. Tel.: 314-362-4185; Fax: 314-747-5354; 2 The abbreviations used are: PLP, pyridoxal phosphate; CPK, Corey-Pauling-Koltun E-mail: [email protected]. model. JANUARY 20, 2006•VOLUME 281•NUMBER 3 JOURNAL OF BIOLOGICAL CHEMISTRY 1305 MINIREVIEW: Enzymes Activated by Mϩ TABLE 1 Classification of M؉-activated enzymes Enzyme Protein Data Bank entrya Ligandsb Ref.