. Author manuscript; available in PMC: 2022 Jan 20.

Published in final edited form as: J Am Chem Soc. 2021 Jan 4;143(2):785–797. doi: 10.1021/jacs.0c09423

Table 1.

Kinetics Parameters of Enolase and Mutants^a

Enzyme	k_cat (s⁻¹)	K_M (μM)	k_cat/K_M (μM⁻¹ s⁻¹)	E_a(k_cat) (kcal mol⁻¹)^b	KIE
WT	82 (5)	17 (3)	3.5	5.7 (0.3)	2.04 (0.13)
Leu343Ile	26 (1)	23 (5)	1.1	10.6 (0.5)	n.d.
Leu343Val	14.4 (0.2)	20 (5)	0.72	8.6 (0.2)	n.d.
Leu343Ala	0.127 (0.005)	8 (1)	0.016	9.3 (0.3)	1.97 (0.07)
Leu343Gly	0.188 (0.002)	23 (5)	0.0082	6.4 (0.3)	n.d.

Initial rates for conversion of 2-PGA to PEP were measured using a continuous spectroscopic assay, in which the formation of PEP is monitored at 240 nm.⁵⁷ All experiments were carried out in 50 mM HEPES, 2 mM MgCl₂ at pH 7.5. The number in parentheses is the standard error. n.d. is not determined. Kinetic parameters k_cat, KIE, and K_M are calculated from experiments at 25°C and are an average of at least three experiments.

E_a values are calculated from k_cat at five different temperatures (10, 20, 25, 30, and 40°C) and are the average of at least three experiments (Table S1, Figure S2).