Kinetic parameters obtained for mutants
| Km | Vmax | |
|---|---|---|
| nM | fmol/min | |
| WT | 330 (267–393) | 101 (95–106) |
| Zn2+ ligands and nearby residues | ||
| D387N | 1475 (670–2280) | 24 (18–31) |
| P388A | >3000 | ND |
| E424Q | 650 (489–812) | 14 (13–16) |
| S454A | 90 (61–119) | 35 (22–37) |
| Y552F | 527 (196–934) | 26 (19–33) |
| Substrate ligands | ||
| R463K | 328 (188–468) | 104 (91–117) |
| R463I† | 1580 (1410–1750) | 90 (83–94) |
| K499E | 193 (117–269) | 118 (108–129) |
| K500R | 471 (267–675) | 142 (121–165) |
| K500E 4-a | ND | ND |
| S501A | 332 (224–440) | 77 (70–83) |
| N519D | ND | ND |
| R536K | 266 (214–318) | 84 (78–89) |
| R536E | >3000 | ND |
| T538V | 1341 (1113–1569) | 99 (90–108) |
| K545R | 272 (250–294) | 132 (129–135) |
| K545E | 1166 (917–1415) | 73 (65–80) |
| Glycosylation | ||
| N459D | 387 (189–586) | 50 (42–58) |
The Michaelis-Menten parameters K m andV max for NAAG were determined by saturation kinetics for mutants and compared with native enzyme wild type (WT), see Fig. 3for examples. V max has been adjusted by the relative intensities of protein expression quantitated by an immunoblot by the ratio of WT to mutant. Italics indicate slightly altered migration observed in an immunoblot.
↵4-a Indicates low level of expression in an immunoblot. ND, indicates not determined. The 95% confidence limits are presented in parentheses.