Its activities for fructose-6-phosphate, glycerol 1-phosphate and phosphoenolpyruvate were about the same and much less than the one for pNPP. Table 5 Kinetic parameters for the activities of C-His-Rv2135c with different substrates at pH 5.8 Specific activity (mol/min/mg) MEK inhibitor Km (mM) p-Nitrophenol Phosphate 0.23 ± 0.07 10.60 ± 0.07 Phosphoenolpyruvate 0.09 ± 0.002 11.25 ± 0.75
Glycerol-1-phosphate 0.05 ± 0.002 14.00 ± 0.00 ADP 0.00 3-Phosphoglyceric acid 0.00 Glucose-6-phosphate 0.00 Fructose-6-phosphate 0.08 ± 0.009 7.75 ± 0.75 Native molecular mass and stability The size of the native form of C-His-Rv2135c was estimated by gel filtration to be 104.70 kDa. With the amino acid calculated size of 25.95 kDa, this suggests that C-His-Rv2135c forms a tetramer in the native state. This conforms to the results obtained by ND-PAGE, which provided the estimated native size of 103.85 kDa. The molecular mass of the native form of C-His-Rv0489 estimated from the gel filtration is 56.02 kDa. This indicates that C-His-Rv0489 forms a dimer, given both calculated and SDS-PAGE estimated molecular mass of the monomer of 28 kDa. The acid phosphatase activity of C-His-Rv2135c at pH 5.8 was found to be enhanced by 15% in the presence
of 10 mM magnesium ion. The enzyme was found to be stable in 50% glycerol at −20°C for up to 4 months with no significant change in activity. Discussion In addition to Rv2419c [17] and Rv3214 [3] characterized recently, we have presented the study of a new mycobacterial Selinexor phosphatase belonging to the histidine phosphatase superfamily. We report the first cloning, expression and characterization of Rv2135c, annotated as hypothetical in the genome database of M. tuberculosis[18]. Simple NCBI BLAST [35, 38] reveals that most of the proteins similar to Rv2135c are annotated as hypothetical proteins or phosphoglycerate mutases. We demonstrated that C-His-Rv2135c possesses neither phosphoglycerate mutase nor phosphoglycerate phosphatase activity. However, it has phosphatase activity in acidic Protein kinase N1 condition. Our findings support the necessity to experimentally characterize enzymes before
their biochemical functions can be ascertained. This is important especially for the histidine phosphatase superfamily whose members can perform different metabolic functions [3, 4, 9, 19]. C-His-Rv2135c has 6 more histidine residues at the C- terminal region than the native protein. The method of C-terminal tagging is commonly used for facilitating purification of enzymes and generally does not affect enzyme specificities. The specific acid phosphatase activity of C-His-Rv2135c (0.23 μmol/min/mg) is about 10 times less than that of Rv3214 (2.6 μmol/min/mg). However, some acid phosphatases of other pathogenic microorganisms are known to possess less specific activities than that of C-His-Rv2135c. Examples include the phosphatases of Francisella tularensis with specific activity of 0.