Extended Data Table 3|.

NAD⁺ kinetic parameters for hUGDH^a

hUGDH	Km (NAD+, mM)	K0.5b (NAD+, mM)	Hill (h)	Kcatc (S-1)	UDX (Ki, μM)	αnadd	# of Data Pointse
FL	-------------	0.8 ± 0.20	0.8 ± 0.1	0.9 ± 0.08	-------------	-------------	18
ΔID	-------------	0.3 ±0.06	0.6 ± 0.1	0.7 ±0.03	-------------	-------------	12
FL-dimer	2.0 ± 0.26	-------------	-------------	0.1 ± 0.01	2.1 ±0.4	0.9 ±0.2	37
ΔID-dimer	3.2 ±0.10	-------------	-------------	0.2 ±0.01	3.6 ±0.8	0.6 ±0.2	47
R1	-------------	0.4 ± 0.03	0.9 ±0.1	0.7 ±0.01	-------------	-------------	17
R2	-------------	0.8 ±0.14	0.7 ± 0.1	0.7 ±0.01	-------------	-------------	15
−Lys	-------------	2.9 ±0.61	0.8 ± 0.1	0.6 ±0.04	-------------	-------------	10
−Pro	-------------	0.5 ±0.06	0.6 ± 0.1	1.2 ±0.03	-------------	-------------	12
0.13×FL	-------------	0.4 ±0.03	0.7 ± 0.1	1.0 ±0.03	-------------	-------------	13
0.26×FL	-------------	0.2 ± 0.03	0.8 ± 0.2	0.8 ±0.05	-------------	-------------	11
0.5×FL	-------------	0.3 ±0.03	0.9 ±0.1	0.9 ±0.02	-------------	-------------	12
2×FL	-------------	1.4 ±0.31	0.8 ±0.1	0.9 ±0.01	-------------	-------------	18
0.13×Ser	-------------	0.9 ±0.24	0.7 ±0.1	1.2 ±0.10	-------------	-------------	12
0.26×Ser	-------------	1.0 ± 0.27	0.7 ± 0.1	1.1 ± 0.09	-------------	-------------	15
0.5×Ser	-------------	1.2 ±0.34	0.7 ± 0.1	1.1 ± 0.09	-------------	-------------	13
Ser		1.3 ± 0.19	0.7 ± 0.1	1.0 ± 0.04	-------------	-------------	13

^aKinetic parameters and associated standard errors (±) for all constructs were derived from global analyses of data in Extended Data Fig. 2

^bHexameric hUGDH displays negative cooperativity with NAD⁺ binding, which indicates a mix of high affinity and low affinity sites^23–28. In previous work, we showed that the native hUGDH_FL K_0.5 of 0.8 mM NAD⁺ corresponds to a mix of high and low affinity sites of (K_M of 88 μM and 1.8 mM, respectively)²³. This is consistent with the published K_d of 30 μM for the coenzyme²⁴.

^cOne catalytic turnover of UDP-GlcA produces two molecules of NADH per cycle

^dα describes the mode of mixed inhibition (Equation 8). An α < 1 in the NAD⁺ saturation curves show that UDP-Xyl binds preferentially to the allosteric binding, and secondarily to the coenzyme binding site.

^eThe number of independent data points used in nonlinear regression (see Methods).