Article Figures & Data
Figures
- Fig. 1.
Nucleotide sequence of the M. morganii PhoC acid phosphatase gene and predicted amino acid sequence. A 747-bp ORF is shown with the deduced 249-amino-acid sequence. Sequences confirmed by protein sequencing are underlined. The signal sequence of the PhoC protein is doubly underlined. The amino acid residues substituted in the course of random mutagenesis are shaded in black. The mutagenic primer is indicated by an arrow. Three phosphatase motif domains are indicated in boxes under the deduced amino acid sequence.
- Fig. 2.
Effect of pH on the synthesis of 5′-IMP. The time course of 5′-IMP synthesis was measured at different pHs with 0.1 M sodium acetate buffer: pH 3.5 (○), pH 4.0 (●), pH 5.0 (□), pH 5.5 (▴), and pH 6.0 (▵). The reaction was carried out at 30°C in a reaction mixture consisting of 0.1 M sodium acetate buffer containing (per liter) 20 g of inosine (74.6 mM), 150 g of disodium hydrogen pyrophosphate (200 mM), and 10 g (dry weight) of E. coli JM109(pMPI501) cells.
- Fig. 3.
5′-IMP synthesis using E. coli overproducing wild-type or mutated acid phosphatases. The time course of 5′-IMP synthesis by resting cells of E. coli JM109(pMPI501) (thin lines), E. coli JM109(pMPI600) (broken lines), and E. coli JM109(pMPI700) (thick lines) was measured. The reaction was carried out at pH 4.0 and 30°C in a reaction mixture consisting of 0.1 M sodium acetate buffer (pH 4.0) containing various concentration of inosine, 150 g of disodium hydrogen pyrophosphate per liter (200 mM), and 10 g (dry weight) of each type of cell per liter. Inosine was added to the reaction mixture at 20 g/liter (74.6 mol/liter) (▴), 40 g/liter (149 mol/liter) (○), and 60 g/liter (224 mol/liter) (●).
Tables
- Table 1.
Kinetic constants for transphosphorylation and dephosphorylation reactionsa
Activity Substrate Strain (plasmid) Km(μM) Vmax (U/mg) Phosphotransferase Inosine Wild type (pMPI501) 117,000 ± 1,000 6.09 ± 0.10 I171T (pMPI600) 73,900 ± 2,800 2.77 ± 0.11 G92D (pMPI502) 114,000 ± 3,000 0.983 ± 0.050 I171T-G92D (pMPI700) 42,600 ± 1,800 2.67 ± 0.11 5′-Nucleotidase 5′-IMP Wild type (pMPI501) 836 ± 26 30.3 ± 0.9 I171T (pMPI600) 1,630 ± 46 11.6 ± 0.4 G92D (pMPI502) 1,490 ± 20 4.70 ± 0.19 I171T-G92D (pMPI700) 1,350 ± 14 5.67 ± 0.15 Acid phosphatase p-NPP Wild type (pMPI501) 41.2 ± 1.0 82.9 ± 1.8 I171T (pMPI600) 64.2 ± 2.1 97.4 ± 3.8 G92D (pMPI502) 116 ± 5 42.3 ± 4.9 I171T-G92D (pMPI700) 50.6 ± 3.1 30.6 ± 1.8 ↵a The enzyme activities were assayed as described in Materials and Methods. The initial velocities were determined, and the steady-state kinetic constants were calculated by using a Lineweaver-Burk plot. Km andVmax values are given as means ± standard deviations.
