Table 3.

Microscopic rate constants for the reactions of the IMPDH mutants. Global fits of the reaction of Scheme 1 were obtained for reaction progress curves for R322A, E323A and Q324A using Dynafit (17). The rate constants of R322E were estimated from the values of k_cat and isotope effects, with the assumption that the intrinsic isotope effects are 3.

Parameter	Wild-typea	R322Ab conform fast	R322Ac conform slow	R322E	E323A	Q324A
NAD+ on, k5 (×10−3 M−1s−1)	29 ± 2	5 ± 1	6 ± 1	≥3.5	300 ± 200	32 ± 6
NAD+ off, k6 (s−1)	4 ± 1	19 ± 3	26 ± 4	≥740 (≥Km/k5)	540 ± 390	140 ± 40
Hydride transfer forward, k7 (s−1)	34 ± 2	7.5 ± 0.9	7.8 ± 0.8	~0.015	16.3 ± 0.4	41 ± 3
Hydride transfer reverse, k8 (s−1)	59 ± 5	5.7 ± 0.8	5.3 ± 0.6	n.d.	5.2 ± 0.4	3.7 ± 0.4
NADH release, k9 (s−1)	8.5 ± 0.4	2.9 ± 0.3	2.9 ± 0.1	fast	12.7 ± 0.5	4.9 ± 0.2
kclose	14000	35 ± 760	1.4 ± 0.3	fast	140 ± 140	30 ± 80
kopen	1000	3 ± 25	n.a.	fast	9 ± 5	6 ± 30
NAD+ inhibit on, k11 (×10−6 M−1s−1)	2	0.1 ± 2	0.06 ± 0.5	n.a.	0.06 ± 0.06	0.01 ± 0.02
NAD+ inhibit off, k12 (s−1)	27 ± 3	20 ± 500	80 ± 600	n.a.	35 ± 46	7 ± 50
kHOH (s−1)	4	1 ± 3	4 (fixed)	0.03	3.4 ± 0.3	0.48 ± 0.01
Calc Kc	n.a.	7	n.a.	n.a.	15	5
sim Km NAD+ (µM) c	110	430	510	n.a.	290	100
sim kcat (s−1) d	2.0	0.3	0.3	n.a.	1.8	0.4
sim Kii NAD+ (µM) c	4300	>5000	>5000	n.a.	>5000	5000
sim Dkcatd,e	1.1	1.1	1.1	n.a.	1.3	1
sim D kcat/Kmd,e	1.2	1.6	1.9	n.a.	2.6	1
sim D2Okcat d,e	2.1	2.1	1.2	n.a.	2.1	2.5
sim D2O kcat/Kmd,e	1.1	0.6	0.6	n.a.	1.1	0.9

^aValues from (9), where the data from (12) was reprocessed using Dynafit. In (12), the values k₅ and k₆ were assigned from reactions with an inactive mutant. However, the mutation perturbed NAD⁺ binding, so we now rely entirely on the global fit to wild-type data. The value of k_HOH was measured experimentally in the reaction with acetlypyridine adenine dinucleotide and fixed during the global fit; the values of k_close and k_open were fixed at aribitrarily fast values such that K_close = 140, and the value of k₁₁ is increased by a factor of (1 + K_c) from (12) to account for the conformational change. Note that while these values are in good agreement with experiment in the value of k_cat and associated isotope effects, they are a rather poor match for isotope effects on k_cat/K_m (compare with Table 1).

^bMechanism assumes that open to closed conformational change is in rapid equilibrium with K_c = 11. The values of k_closed, k_open, k₁₂, are unconstrained- equally good fits are obtained as long as these values are fast relative to the other parameters.

^cMechanism assumes that closing of the flap is partially rate-limiting. The values of k_open and k_HOH are unconstrained by the data, and equally good fits are obtained as long as k_open ≤ 0.1 and k_HOH ≥ 4.

^dThe reactions were simulated using the parameters above, saturating IMP and [NAD⁺] = 25, 50, 100, 250, 500, 1000 and 2000 µM. The resulting initial velocities were fit to Michaelis-Menten with or without NAD⁺ substrate inhibition term to derive the values of k_cat, K_m NAD⁺ and K_ii NAD⁺.

^eAssumes intrinsic isotope effects = 3, no changes in other steps; note that significant inverse solvent isotope effects are predicted on k_cat/K_m because more E-XMP* accumulates in the presence of D₂O, which in turn is trapped by NAD⁺ in E-XMP*•NAD⁺, decreasing the value of K_m.