Table 3. Isotope Effects on the Arrhenius Parameters^a of the Hydride Transfer of l- and h-DHFR in the Temperature Range 5–45 °C.

enzyme	l-DHFR	h-DHFR
temp, °C	5–45	25–45	5–25
AH/AT	7.0 ± 1.5b	5.6 ± 1.9	0.01 ± 0.003d
ΔEa,H-T (kcal/mol)	–0.1 ± 0.2b	0.1 ± 0.2	4.1 ± 0.3
DAD (Å)c	3.06 ± 0.08	3.06 ± 0.0008	3.36 ± 0.01

^aThe values of these parameters are obtained by fitting temperature dependence of ^Tk_hyd to eq 2, measured by competitive experiments at pH 9 (Figure 4).

^bData from ref (31).

^cDAD is the average distance between C4 of NADPH and C6 of DHF in the dominant population at the TRS. This parameter is estimated from fitting KIE_int to a Marcus-like model following the method published in ref (35).

^dThe steeply temperature-dependent KIEs and small ratio of pre-exponential factors (A_H/A_T) can be interpreted either by the semiclassical TS theory corrected with moderate tunneling,³⁴ or by a Marcus-like model with multiple populations with different DADs.³⁵