Purification of polyphosphate and ATP glucose phosphotransferase from Mycobacterium tuberculosis H37Ra: evidence that poly(P) and ATP glucokinase activities are catalyzed by the same enzyme.
Polyphosphate [poly(P)n]:D-(+)-glucose-6-phosphotransferase (EC 2.7.1.63) from Mycobacterium tuberculosis H37Ra was purified to homogeneity using an improved method which yielded a 634-fold purification with higher recovery. The purified enzyme migrated as a single band with M(r) 33 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The native enzyme was shown to be a dimer by gel filtration using high-performance liquid chromatography (HPLC). The purified enzyme fractionated as a single peak on a C8 reverse-phase HPLC column and was found to display both polyphosphate- and ATP-dependent glucokinase activities. Further evidence that a single protein was responsible for both activities was shown by nondenaturing PAGE, in which the two activities (as determined by an activity stain in dual experiments) were found to comigrate. The C-terminal analysis yielded a single sequence while the N-terminus which was blocked also yielded a single sequence after deblocking. The two activities were found to have the same temperature optimum of 50 degrees C. The pH optima were 9.5 and 8.6-9.5 with poly(P)32 and ATP as the phosphoryl donors, respectively. The apparent Km for poly(P)32 was 18.4 microM while the Km for ATP was 1.46 mM. In addition, the nucleotide analogue, Reactive Blue 4, was found to be a competitive inhibitor with ATP in the ATP-dependent glucokinase reaction, while it displayed noncompetitive inhibition patterns with poly(P) in the poly(P)-dependent glucokinase reaction. It is concluded that the poly(P) and ATP glucokinase activities are catalyzed by the same enzyme but that the two substrates may have different binding sites.
Hsieh
P C
PC
Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106.
Shenoy
B C
BC
Jentoft
J E
JE
Phillips
N F
NF
D000595:Amino Acid Sequence; D002384:Catalysis; D002853:Chromatography, Liquid; D004396:Coloring Agents / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005941:Glucokinase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D010770:Phosphotransferases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011122:Polyphosphates / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity; D013696:Temperature; D014227:Triazines / Q000494:pharmacology
D004396:Coloring Agents; D011122:Polyphosphates; D014227:Triazines; C036598:procion blue MX-R; D010770:Phosphotransferases; D005941:Glucokinase