SCIENCE & TECHNOLOGY
IDENTITY OF MYSTERY ATOMS DISPUTED Contested crystallographic assignment raises questions about enzymatic phosphoryl transfer AMANDA YARNELL, C&EN WASHINGTON
parently, it was stabilized sufficiently to be study that reported the trapobserved in the enzyme. Allen and Dunping of a high-energy species away-Mariano chalked this up to the enin an enzyme transferring a zyme's ability to minimize entropy in the phosphoryl group (P0 3 2 ~) to enzyme-substrate complex and make faits substrate seemed to answer fundamenvorable hydrogen bonds to the two apical tal questions about how enzymes catalyze oxygens. Other scientists have suggested the transfer of phosphoryl groups. But the that the extremely low temperature used chemical assignment of this species has for the crystallographic studies may have now been questioned—once again openplayed a role in trapping this species. ing the issue of how enzymes carry out this But a group of British scientists think critical biochemical reaction. that these factors can't possibly stabilize In March, crystallographer Karen N . such a high-energy species and that there Allen of Boston University School of Medmay be another explanation. Chemists G. icine and biological chemistry professor DeMichael Blackburn and Nicholas H. Wilbra Dunaway-Mariano of the University of liams ofSheffield University in England, and New Mexico reported that they had usedXstructural biologists StevenJ. Gamblin and ray crystallography performed at -180 °C to Stephen J. Smerdon of London's National catch an enzyme in the act of transferring a phosphoryl group to one of its natural substrates [Science, 2 9 9 , 2067 CONTROVERSIAL The identity of (2003); C&EN, March 31, the trigonal bipyramidal species found page 5}. In the active site of in the active site of a phosphoryltheir 1.2-A resolution transfer enzyme has come into question. Although it structure of the enzyme was originally identified phosphogjucomutase, •A as oxyphosphorane, Allen and Dunawaysome have suggested Mariano identified a ' that it might actually be trigonal bipyramidal oxy- % magnesium trifluoride phosphorane species suspend(phosphorus, purple; oxygen, ed between the enzyme and its red; carbon, yellow). glucose substrate. With P-O bond lengths that clearly correspond
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to a sig^ W ' nificant degree of bonding, this species led the authors to suggest that enzymes catalyze the transfer of ^- / phosphoryl groups via an associative nucleophilic substitution mechanism. The trigonal bipyramidal oxyphosphorane species should be high-energy and intrinsically unstable—and therefore it was widely expected to be impossible to catch it by crystallography. But somehow, ap30
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InstituteforMedical Research (NIMR) recendy suggested that what's in the active site might not be an oxyphosphorane after all [Science, 301,1184 (2003)1. "It's far more likely that what's been observed in the active site of the phosphoglucomutase structure is magnesium trifluoride," Blackburn says. Blackburn and his coworkers have based their alternate assignment on a 1.8-A resolution crystal structure of a small G-pro-
tein complexed with its activating protein [Chem. Biol., 9,375 (2002)1. In this structure—which was investigated because the interaction of these two proteins is dependent on both magnesium and fluoride—Smerdon, Gamblin, and their N I M R colleague John F. Eccleston found what they believe to be a magnesium trifluoride ion strung between an oxygen atom on the enzyme and another from an enzyme-bound water molecule. They based their assignment on a measurement of the ratio of phosphorus to magnesium atoms via a spectroscopic technique called proton-inducedX-ray emission.This technique can be used to measure the ratios of two elements with atomic numbers greater than 11—but it gives no spatial information. In that case, crystals were grown in the presence of 10 m M Mg 2 + and 10 m M NH 4 F. Allen and Dunaway-Mariano used 10 mM Mg2* and 100 mM N H 4 F in their phosphoglucomutase experiments, leading Blackburn and his colleagues to conclude that the trigonal bipyramidal species in that enzyme structure might be magnesium trifluoride, too. But Allen and Dunaway-Mariano are sticking to their original assignment [Science, 301,1184 (2003)1. Among other evidence, they point to an anomalous scattering experiment that shows that the magnitude of the electron density of the central atom of the oxyphosphorane is identical to that of the other, uncontested phosphorus atom in the structure. Because magnesium contains fewer anomalously scattering electrons than phosphorus, this suggests that both atoms are indeed phosphorus. Allen and Dunaway-Mariano also note that their phosphoglucomutase crystals contain the expected number of phosphorus atoms per molecule of enzyme. Plus they report that 3 m M N H 4 F does not inhibit enzyme catalysis—even though they can grow crystals in solutions containing as little as lmMNH 4 F—implying that F does not end up in the active site. BLACKBURN ADMITS that magnesium trifluoride is unlikely to form in solution in readily detectable amounts under such low concentrations of fluoride ion. Nor is it likely to form under the previously published conditions for crystallizing phosphoglucomutase (100 mM NH 4 F) or the small G-protein (10 mM NH 4 F). Rather, he tells C&EN, the enzyme active site must somehow preferentially stabilize this species. If the trigonal bipyramidal species is indeed an oxyphosphorane—the enzyme caught in the act of catalysis with its true HTTP://WWW.CEN-ONLINE.ORG
substrate—"it demands a reevaluation of much ofour thinking on phosphoryl-transfer mechanisms in enzymes," Blackburn says.
an associative or dissociative transition state, because that's the species that the enzyme is stabilizing. He points out that phosphoryl-transfer reactions can have transition-state structures that run the gamut, from dissociative ones (in which the P-O bond to the leaving group is nearly broken and the P-O bond to the incoming nucleophile is barely formed) to associative ones (in which the P-O bond to the incoming nucleophile is almost fully formed and the P-O bond to the leaving group is nearly cleaved). Allen and Dunaway-Mariano "note that the oxyphosphorane's bond lengths are longer than would be expected for a fully formed P-O bond," Herschlag points out. Assuming it's really an oxyphosphorane that they are observing, "the P-O bonding they observe is what would be expected for a dissociative transition state with metaphosphate character," he argues. The same kind of mechanism is observed in uncatalyzed phosphoryl-transfer reactions in aqueous solution, he tells C&EN. But if what's in the central position of the trigonal bipyramidal species turns out to be magnesium and not phosphorus,
ENZYMOLOGISTS have long puzzled over whether the mechanism is SNl-like, SN2like, or addition-elimination. The trigonal bipyramidal species' apical bond lengths are too short for it to be a metaphosphate species "in-flight" as part of a fully dissociative, SNl-like reaction. Blackburn's colleague Williams tells C&EN that the bond lengths and bond orders observed in the putative oxyphosphorane species would suggest that phosphoryl transfer occurs through an addition-elimination process. He thinks that the simple fact that an intermediate is observed at all seems to suggest that the reaction can't be SN2-like. This argument alone, however, may not rule out a S^-like mechanism because under different conditions (such as when the temperature is raised) it's quite possible that the intermediate won't be observed. According to biochemist Daniel Herschlag ofStanford University, the real question is whether the reaction proceeds via
"this would be one ofmany transition-state analogs that have been reported over the years," Herschlag says. 'An analog will rearrange to its most stable state in the enzyme's active site, which may or may not reflect the true transition state," he adds, noting that because of this, a magnesium trifluoride transition-state analog would not provide direct information about the structure of the transition state. Albert S. Mildvan, abiochemist atJohns Hopkins School ofMedicine, remarks that it remains to be seen who is right. What is needed, he points out, are more experiments to test whether the trigonal bipyramidal intermediate can be detected in the absence of fluoride and whether fluoride is apotent inhibitor ofphosphoglucomutase. More direct solid-state 31P and perhaps 19F nuclear magnetic resonance spectroscopy experiments may also be useful. Structural biologist David E. Wemmer of the University of California, Berkeley, agrees that the jury is still out on this one. He tells C&EN that he finds the British team's alternate interpretation "intriguing." But itll take more experiments "to sort out which picture is really correct," he adds. •
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