Table 2.

Summary of Amino Acid Substitutions Made to the Putative rS-HPCDH35 Catalytic Residues Using Site-Directed Mutagenesis ^a

amino acid	postulated catalytic role(s)b	substitution	Km (μM S-HPC)	kcat (s-1)	kcat/Km (M-1 s-1)
		none	31 ± 0.9	25 ± 0.2	7.5 × 105
S143	H-bond donor to substrate/charge stabilization of the transition state	S143A	9500 ± 2500	0.013 ± 0.001	1.4 × 100
Y156	general acid/base	Y156A	1800 ± 200	0.65 ± 0.02	3.5 × 102
		Y156F	no activityc
K160	lowers pKa of Y156/coenzyme binding	K160A	no activityc

^aAssays contained 66 μg of S143A or 20 μg of Y156A, 5 mM NAD+ and variable concentration of S-HPC.

^bPostulated catalytic roles are based on a general trend found in most SDR enzymes (including R-HPCDH).

^cNo activity was defined as no measurable change in the absorbance at 340 nm (for NADH) in assays containing 100 μg protein, 2.5 mM S-HPC and 20 mM NAD⁺. Apparent k_cat and K_m values are reported as means ± standard errors. All other values are reported as means only. All assays were performed in triplicate at 30 °C with fixed concentrations of NAD⁺. Apparent kinetic constants were determined by fitting experimental data to the standard form of the Michaelis-Menten equation.