Disruption of NAD+ binding site in glyceraldehyde 3-phosphate dehydrogenase affects its intranuclear interactions

Table 1 Mutations introduced to glyceraldehyde 3-phosphate dehydrogenase cDNA ORF

GAPDH variant	Mutagenic primers1	Nucleotide change	Codon change
Y94A	CGATGCTGGCGCTGAGGCCGTCGTGGAGTCCACTGG	280-281	UAC- > GCC
	CCAGTGGACTCCACGACGGCCTCAGCGCCAGCATCG
S98A	GAGTACGTCGTGGAGGCCACTGGCGTCTTCACC	290	UCC- > GCC
	GGTGAAGACGCCAGTGGCCTCCACGACGTACTC
T99A	AGTACGTCGTGGAGTCCGCTGGCGTCTTCACCACC	295	ACU- > GCU
	GGTGGTGAAGACGCCAGCGGACTCCACGACGTACT
T99I	GAGTACGTCGTGGAGTCCATTGGCGTCTTCA	296	ACU- > AUU
	TGAAGACGCCAATGGACTCCACGACGTACTC

¹Mutated nucleotides are indicated in bold font; GAPDH: Glyceraldehyde 3-phosphate dehydrogenase.

Table 2 Kinetic parameters of wild type and mutated glyceraldehyde 3-phosphate dehydrogenase variants

GAPDH variant	Km (G3P) (μmol/L)	Km (NAD+) (μmol/L)
Wild type	77 ± 25.9	57 ± 11.1
Y94A	87 ± 33.1	97.4 ± 11
S98A	100 ± 36.7	101 ± 7.2
T99A	112 ± 44.7	105 ± 17.7
T99I	106 ± 38.3	741 ± 257

GAPDH: Glyceraldehyde 3-phosphate dehydrogenase.

Table 3 Fluorescence recovery after photobleaching analysis of dynamic properties of wild type and mutated enhanced green fluorescent protein-glyceraldehyde 3-phosphate dehydrogenase

Protein	Compartment	(1-Mf)	t1/2, s	D,μm2/s
EGFP-GAPDH-WT	cyto	0.18 ± 0.038	0.19 ± 0.055	27.17 ± 1.632
EGFP-GAPDH-Y94A	cyto	0.17 ± 0.129	0.09 ± 0.014	43.76 ± 7.158
EGFP-GAPDH-S98A	cyto	0.20 ± 0.009	0.12 ± 0.016	29.15 ± 3.885
EGFP-GAPDH-T99A	cyto	0.11 ± 0.062	0.11 ± 0.023	35.52 ± 8.133
EGFP-GAPDH-T99I	cyto	0.17 ± 0.06	0.22 ± 0.041	17.46 ± 1.733
EGFP-GAPDH-WT	nuclei	0.62 ± 0.048	1.41 ± 0.033	2.91 ± 0.331
EGFP-GAPDH-Y94A	nuclei	0.23 ± 0.127	0.09 ± 0.034	39.05 ± 6.291
EGFP-GAPDH-S98A	nuclei	0.35 ± 0.136	0.13 ± 0.018	26.57 ± 3.747
EGFP-GAPDH-T99A	nuclei	0.38 ± 0.120	0.11 ± 0.027	33.01 ± 9.209
EGFP-GAPDH-T99I	nuclei	0.29 ± 0.028	0.21 ± 0.059	14.97 ± 1.855

Cyto: Cytoplasmic compartment; nuclei: Nuclear compartment; EGFP: Enhanced green fluorescent protein; GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; FRAP: Fluorescence recovery after photobleaching.

Table 4 Average potential energy, kinetic energy, total energy and temperature calculated from dynamic simulations

Polypeptide variant	Average potential (kcal)	Average kinetic energy (kcal)	Average total energy (kcal)	Average temperature (°C)
T99	-5021.8	97.1	-4924.7	293.4
A99	-5007.3	93.7	-4913.6	293.7
I99	-4997.8	101.4	-4896.4	293.3