Kinetic measurements of phosphoglucose isomerase and phosphomannose isomerase by direct analysis of phosphorylated aldose–ketose isomers using tandem mass spectrometry

Abstract A mass spectrometry based method for the direct determination of kinetic constants for phosphoglucose isomerase (PGI) and phosphomannose isomerase (PMI) is described. PGI catalyzes the interconversion between glucose-6-phosphate (Glc6P) and fructose-6-phosphate (Fru6P) and PMI performs the same function between mannose-6-phosphate (Man6P) and Fru6P. These two enzymes are essential in the pathways of glycolytic or oxidative metabolism of carbohydrates and have been considered as potential therapeutic targets. Traditionally, they are assayed either by spectrophotometric detection of Glc6P with one or more coupling enzymes or by a colorimetric detection of Fru6P. However, no suitable assay for Man6P has been developed yet to study the reaction of PMI in the direction from Fru6P to Man6P. In the work presented herein, a general assay for the isomeric substrate-product pair between Glc6P and Fru6P or between Man6P and Fru6P was developed, with the aim of directly studying the kinetics of PGI and PMI in both directions. The 6-phosphorylated aldose and ketose isomers were distinguished based on their corresponding tandem mass spectra (MS 2 ) obtained on a quadrupole ion trap mass spectrometer, and a multicomponent quantification method was utilized to determine the composition of binary mixtures. Using this method, the conversion between Fru6P and Glc6P and that between Fru6P and Man6P are directly monitored. The equilibrium constants for the reversible reactions catalyzed by PGI and PMI are measured to be 0.3 and 1.1, respectively, and the kinetic parameters for both substrates of PGI and PMI are also determined. The values of K M and V max for Fru6P as substrate of PMI are reported to be 0.15 mM and 7.78 μmol/(min mg), respectively. All other kinetic parameters measured correlate well with those obtained using traditional methods, demonstrating the accuracy and reliability of this assay.

[1]  J. Leary,et al.  Compositional analysis and quantification of heparin and heparan sulfate by electrospray ionization ion trap mass spectrometry. , 2003, Analytical chemistry.

[2]  G. Paravicini,et al.  PMI40, an intron-containing gene required for early steps in yeast mannosylation , 1992, Molecular and cellular biology.

[3]  Yonghao Yu,et al.  Characterization of noncovalent protein-ligand complexes and associated enzyme intermediates of GlcNAc-6-O-sulfotransferase by electrospray ionization FT-ICR mass spectrometry , 2004, Journal of the American Society for Mass Spectrometry.

[4]  H. Freeze,et al.  Congenital Disorders of Glycosylation: Glycosylation Defects in Man and Biological Models for Their Study , 2001, Biological chemistry.

[5]  Bruce W. Lee,et al.  Phosphomannoisomerase and phosphoglucoisomerase in seeds of cassia coluteoides and some other legumes that synthesize galactomannan , 1984 .

[6]  P. Orlean.  Dolichol phosphate mannose synthase is required in vivo for glycosyl phosphatidylinositol membrane anchoring, O mannosylation, and N glycosylation of protein in Saccharomyces cerevisiae , 1990, Molecular and cellular biology.

[7]  J. Leary,et al.  Multiplex inhibitor screening and kinetic constant determinations for yeast hexokinase using mass spectrometry based assays , 2003, Journal of the American Society for Mass Spectrometry.

[8]  O. H. Lowry,et al.  The kinetics of phosphoglucoisomerase. , 1960, The Journal of biological chemistry.

[9]  K. Röhm,et al.  Glucose‐6‐phosphate isomerase from ehrlich ascites tumor cells: Purification and some properties , 1973, FEBS letters.

[10]  M. Gelb,et al.  Affinity capture and elution/electrospray ionization mass spectrometry assay of phosphomannomutase and phosphomannose isomerase for the multiplex analysis of congenital disorders of glycosylation types Ia and Ib. , 2003, Analytical chemistry.

[11]  H. Desaire,et al.  A strategy for the determination of enzyme kinetics using electrospray ionization with an ion trap mass spectrometer. , 2001, Analytical chemistry.

[12]  K. Brand,et al.  The diagnostic validity of the serum tumor marker phosphohexose isomerase (PHI) in patients with gastrointestinal, kidney, and breast cancer. , 1990, Cancer investigation.

[13]  A. Zanella,et al.  Study of the molecular defects in glucose phosphate isomerase-deficient patients affected by chronic hemolytic anemia. , 1996, Blood.

[14]  J. Leary,et al.  Determination of enzyme/substrate specificity constants using a multiple substrate ESI-MS assay , 2004, Journal of the American Society for Mass Spectrometry.

[15]  R. Hubbard,et al.  The X-ray crystal structure of phosphomannose isomerase from Candida albicans at 1.7 Å resolution , 1996, Nature Structural Biology.

[16]  C. Petzold,et al.  Investigation of ion/molecule reactions as a quantification method for phosphorylated positional isomers: An FT-ICR approach , 2003, Journal of the American Society for Mass Spectrometry.

[17]  Selenomethionine labelling of phosphomannose isomerase changes its kinetic properties. , 1995, European journal of biochemistry.

[18]  F. Schneider,et al.  Kinetik der Glucosephosphat-Isomerase (EC 5.3.1.9) aus Hefe in vitro und ihre Anwendung auf Flußberechnungen durch die Gärungskette der anaeroben Hefezelle , 1970 .

[19]  Heather Desaire,et al.  Detection and quantification of the sulfated disaccharides in chondroitin sulfate by electrospray tandem mass spectrometry , 2000, Journal of the American Society for Mass Spectrometry.

[20]  Leishmania mexicana mexicana glucose-6-phosphate isomerase: crystallization, molecular-replacement solution and inhibition. , 2004, Acta crystallographica. Section D, Biological crystallography.

[21]  F. Opperdoes,et al.  Subcellular distribution and characterization of glucosephosphate isomerase in Leishmania mexicana mexicana. , 1994, Molecular and biochemical parasitology.

[22]  M. W. Slein.  [37] Phosphohexoisomerases from muscle , 1955 .

[23]  H. Freeze,et al.  Carbohydrate-deficient glycoprotein syndrome type Ib. Phosphomannose isomerase deficiency and mannose therapy. , 1998, The Journal of clinical investigation.

[24]  C. Walsh,et al.  Mass spectrometric characterization of a three-enzyme tandem reaction for assembly and modification of the novobiocin skeleton. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[25]  E. Bertini,et al.  Phosphomannose isomerase deficiency: a carbohydrate-deficient glycoprotein syndrome with hepatic-intestinal presentation. , 1998, American journal of human genetics.

[26]  A. Proudfoot,et al.  Purification and characterization of fungal and mammalian phosphomannose isomerases , 1994, Journal of protein chemistry.

[27]  J. Leary,et al.  Kinetic measurements of phosphoglucomutase by direct analysis of glucose-1-phosphate and glucose-6-phosphate using ion/molecule reactions and Fourier transform ion cyclotron resonance mass spectrometry. , 2004, Analytical biochemistry.

[28]  J. Dyson,et al.  A rapid pH-stat assay for phosphofructokinase and the forward reaction of phosphoglucose isomerase. , 1965, Analytical biochemistry.

[29]  H. Desaire,et al.  Differentiation of diastereomeric N-acetylhexosamine monosaccharides using ion trap tandem mass spectrometry. , 1999, Analytical chemistry.

[30]  C. Jeffery,et al.  Inhibition of type I and type II phosphomannose isomerases by the reaction intermediate analogue 5-phospho-D-arabinonohydroxamic acid supports a catalytic role for the metal cofactor. , 2004, Biochemistry.

[31]  R. W. Gracy,et al.  Studies on phosphomannose isomerase. 3. A mechanism for catalysis and for the role of zinc in the enzymatic and the nonenzymatic isomerization. , 1968, The Journal of biological chemistry.

[32]  J. S. Miles,et al.  Nucleotide sequence and transcriptional start point of the phosphomannose isomerase gene (manA) of Escherichia coli. , 1984, Gene.

[33]  H. Desaire,et al.  Multicomponent quantification of diastereomeric hexosamine monosaccharides using ion trap tandem mass spectrometry. , 1999, Analytical chemistry.

[34]  François,et al.  Improved protocols for quantitative determination of metabolites from biological samples using high performance ionic-exchange chromatography with conductimetric and pulsed amperometric detection. , 2000, Enzyme and microbial technology.

[35]  F. Opperdoes,et al.  Competitive Inhibition of Trypanosoma Brucei Phosphoglucose Isomerase by D-Arabinose-5-Phosphate Derivatives , 2000, Journal of enzyme inhibition.

[36]  J. Chirgwin,et al.  The crystal structure of human phosphoglucose isomerase at 1.6 A resolution: implications for catalytic mechanism, cytokine activity and haemolytic anaemia. , 2001, Journal of molecular biology.

[37]  R. Alberty,et al.  Studies of the Enzyme Fumarase. VII.1 Series Solutions of Integrated Rate Equations for Irreversible and Reversible Michaelis-Menten Mechanisms2 , 1957 .

[38]  M. Rheinnecker,et al.  A novel Saccharomyces cerevisiae secretory mutant possesses a thermolabile phosphomannose isomerase , 1991, Journal of bacteriology.

[39]  C. Bertozzi,et al.  Kinetic analysis of NodST sulfotransferase using an electrospray ionization mass spectrometry assay. , 2002, Biochemistry.

[40]  R. W. Gracy,et al.  Molecular basis for the isozymes of bovine glucose-6-phosphate isomerase. , 1986, Archives of biochemistry and biophysics.

[41]  A. Sols,et al.  SPONTANEOUS AND ENZYMATICALLY CATALYZED ANOMERIZATION OF GLUCOSE 6-PHOSPHATE AND ANOMERIC SPECIFICITY OF RELATED ENZYMES. , 1965, The Journal of biological chemistry.