The disappearance of the substrate and the appearance of the products of the nonenzymatic and K12G TIM-catalyzed reactions of GAP in D

_{2}O was monitored by

^{1}H NMR spectroscopy, as described in previous work (). shows the time course for the disappearance of GAP (10 mM) catalyzed by 85 µM K12G TIM in D

_{2}O at pD 7.9 (10 mM imidazole), 25 °C and

*I* = 0.15 (NaCl). The solid line shows the nonlinear least-squares fit of the experimental data to a single exponential which gave

*k*_{obsd} = 3.6 × 10

^{−4} s

^{−1} as the observed rate constant for disappearance of GAP (). shows the time dependence of the

*observed* fractional yields of the four products of this reaction, (

*f*_{P})

_{obsd} (P = DHAP,

*d*-DHAP,

*d*-GAP or MG). These fractional yields were calculated from the fraction of the particular product P (see Materials and Methods) and the sum of the fractions of all the products of both the enzymatic and nonenzymatic reactions of GAP using (). There is no significant change in (

*f*_{P})

_{obsd} for DHAP with time, but the changes with time in the observed fractional yields of

*d*-DHAP (increasing) and

*d*-GAP (decreasing) result from enzyme-catalyzed isomerization of

*d*-GAP to give the thermodynamically favored product

*d*-DHAP (). reports the

*initial* fractional product yields, (

*f*_{P})

_{o} for DHAP,

*d*-DHAP and

*d*-GAP, or (

*f*_{MG})

_{tot} for MG, that were determined by extrapolation of the observed product yields (

*f*_{P})

_{obsd} to zero time (intercepts in ).(3)

(4)

(5)

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