NOWAK, MILDVAN,
AND
KENYON
Nuclear Relaxation and Kinetic Studies of the Role of Mn2+ in the Mechanism of Enolase? Thomas Nowak,* Albert S. Mildvan, and George L. Kenyon$
When Mn?+ is bound at the active site of enolase, two rapidly exchanging water molecules are coordinated as indicated by the effect of the enolase-Mn2+ complex on the longitudinal relaxation rate (l/T1)of water protons determined a t six frequencies. The exchange rate ( l / r > ~of ) these water molecules is greater than 7.7 X lo6 sec-I, and the correlation sec, a value time for the Mn’--water interaction is 2.2 x which is shown to be dominated by rTs,the electron spin relaxation time of Mn?-. Ternary complexes of the substrates (phosphoenolpyruvate and a-(dihydroxyphosphinylmethy1)acrylate (CH,-PEP)) and inactive substrate analogs (Dphospholactate, L-phospholactate, and phosphoglycolate) all affect l/TI of water protons two-three times less than the binary enoIase-Mn*’ complex at 24.3 MHz. From UTl and l/T, values of the carbon-bound protons of CHn-PEP at two frequencies and of the phosphorus at one frequency, the correlation times ( r C in ) the active ternary CH?-PEP complex are essentially unchanged from that of the binary complex. Similar r, values are obtained by measurements of l/Tl and l/T, of the protons and phosphorus of the inactive analogs. indicating that the decrease in l/Tl of water in the ternary complexes is not due to a change in 7,. The distances from the enzyme-bound Mn2- to the carbon-bound protons ( 6 . 5 ~ ~ 7.5 A) and the phosphorus atoms (5.5-6.2 A) of the active and inactive analogs are too great for inner sphere complexes, but are consistent with second sphere complexes in which a water molecule intervenes between the bound Mn?+ and the bound substrate analog. From the rc values of the analogs and l/Tl of water in the ternary complexes it is calculated that 0.3-1 .O rapidly exchanging water molecule remains coordinated to MnZ- in the various ternary complexes. Hence, the binding of the substrate may have “immobilized” a water ABSTRACT:
E
nolase (phosphoenolpyruvate hydratase, or 2-phosphoD-glycerate hydrolase, E C 4.2.1.1 1) catalyzes the reversible hydration of phosphoenolpyruvate (P-eno1pyruvate)l to 2-
t F r o m the Institute for Cancer Research, Fox Chase, Philadelphia, Pennsylvania 191 11, and the Department of Chemistry, University of California, Berkeley, California 94720. Receitied August 22, 1972. This work was supported by U. S. Public Health Service Grants A3113351, CA-06927, a n d RR-00542 (for use of nmr equipment located a t the University of Pennsylvania), G r a n t RR-05539 from the National Institutes of Health, G r a n t GB-27739X (previously Grant GB-8579) from the National Science Foundation, and by a n appropriation from the Commonwealth of Pennsylvania (T. N. a n d A . S . M.), and by U . S. Public Health Service Grant AM-13529 from the National Institutes of Health (G. L. I19.4 1.99
f(Tc) From l / Tl(HnO)d From
WI
0 . 7 8 1.21'-1.96' 0 . 7 8 1.72'-1.81' 0 . 7 8 1.64'-2.03' 0.78 1.54
x
lo9 sec
From Tl/T2
From l / Tl(H20)d
1.76 1.72 1.45
