CATALYTIC METAL-ION BINDING IN ENOLASE - THE CRYSTAL-STRUCTURE OF AN ENOLASE-MN2-PHOSPHONOACETOHYDROXAMATE COMPLEX AT 2.4-ANGSTROM RESOLUTION()

Enolase, a glycolytic enzyme that catalyzes the dehydration of 2-phosp ho-D-glycerate (PGA) to form phosphoenolpyruvate (PEP), requires two d ivalent metal ions per active site for activity. The first metal ion, traditionally referred to as ''conformational'', binds in a high-affin ity site I. The second metal ion, ''catalytic'', binds in site II only in the presence of a substrate or substrate analogue and with much lo wer affinity for the physiological cofactor Mg2+. While the high-affin ity site has been well characterized, the position of the lower affini ty site has not been established so far. Here, we report the structure of the quaternary complex between enolase, the transition-state analo gue phosphonoacetohydroxamate (PhAH), and two Mn2+ ions. The structure has been refined by using 16 561 reflections with F/sigma a(F) greate r than or equal to 3 to an R = 0.165 with average deviations of bond l engths and bond angles from ideal values of 0.013 Angstrom and 3.1 deg rees, respectively. The ''catalytic'' metal ion is coordinated to two oxygen atoms of the phosphono moiety of PhAH and to the carbonyl oxyge n of Gly37. Most likely, disordered water molecules complement its coo rdination sphere. The interaction with the site II metal ion must stab ilize negative charge on the phosphate group and produce electron with drawal from carbon 2 of the substrate, facilitating proton abstraction from carbon 2, the rate-limiting step in the catalytic process. The G ly37 residue is located in the flexible loop Ser36-His43, which assume s an ''open'' conformation in the absence of substrate and a ''closed' ' conformation in the presence of a substrate. The metal ion binding i n site II must stabilize the ''closed'' conformation and the substrate /product binding. Thus the inhibitory effect of higher Mg2+ concentrat ions on enolase activity may be explained by the sequential reaction m echanism in which the site II metal ion must leave before the product is released from the enzyme.