Table 1.
Enzymatic properties of NPP1, NPP2 and NPP6
| Characteristics | NPP1 | NPP2 | NPP6 |
|---|---|---|---|
| Molecular sizes | |||
| SDS–PAGE (gel filtration) | 70 kDa (251 kDa) | 71 kDa (374 kDa) | 74 kDa (224 kDa) |
| Substrate specificity relative value, %) | ADP-glucose (100) | UDP (100) | ADP (100) |
| ADP-ribose (99) | ADP (87) | UDP (68) | |
| TDP-glucose (82) | APS (81) | ADP-glucose (65) | |
| ATP (81) | CDP (56) | PPi (59) | |
| UDP-glucose (77) | IDP (48) | Bis-p-NPP (35) | |
| CDP-glucose (77) | TDP (40) | APS (31) | |
| GDP-mannose (76) | GDP (38) | TDP (27) | |
| APS (44) | Bis-p-NPP (32) | UDP-glucose (25) | |
| ADP (40) | ADP-glucose (NQa) | TDP-glucose (15) | |
| Optimum pH | pH 6.0 (ADP-glucose) | pH 4.5 (ADP) | pH 4.0 (ADP) |
| pH 5.0 (ADP-glucose) | |||
| Optimum temperature | 60°C | 60°C | 55°C |
| Metal ion requirement | None | None | None |
The molecular sizes of purified NPP1, NPP2 and NPP6 proteins were estimated by SDS–PAGE and gel filtration chromatography with molecular weight standards as described in the Materials and Methods. The data presented in Table 2 are used to summarize substrate specificity. The maximal value of kcat/Km for each NPP enzyme was normalized at 100%. The temperature and pH dependency of the enzyme reaction were determined using ADP-glucose and ADP as substrates. Details of temperature and pH dependency are presented in Supplementary Fig. S2.
APS, adenosine 5′-phosphosulfate; Bis-p-NPP, bis(p-nitrophenyl) phosphate; NQ, not quantifiable; PPi, pyrophosphate.