The reaction mechanism of type I phosphomannose isomerases: new information from inhibition and polarizable molecular mechanics studies.
Type I phosphomannose isomerases (PMIs) are zinc-dependent metalloenzymes involved in the reversible isomerization of D-mannose 6-phosphate (M6P) and D-fructose 6-phosphate (F6P). 5-Phospho-D-arabinonohydroxamic acid (5PAH), an inhibitor endowed with nanomolar affinity for yeast (Type I) and Pseudomonas aeruginosa (Type II) PMIs (Roux et al., Biochemistry 2004; 43:2926-2934), strongly inhibits human (Type I) PMI (for which we report an improved expression and purification procedure), as well as Escherichia coli (Type I) PMI. Its K(i) value of 41 nM for human PMI is the lowest value ever reported for an inhibitor of PMI. 5-Phospho-D-arabinonhydrazide, a neutral analogue of the reaction intermediate 1,2-cis-enediol, is about 15 times less efficient at inhibiting both enzymes, in accord with the anionic nature of the postulated high-energy reaction intermediate. Using the polarizable molecular mechanics, sum of interactions between fragments ab initio computed (SIBFA) procedure, computed structures of the complexes between Candida albicans (Type I) PMI and the cyclic substrate β-D-mannopyranose 6-phosphate (β-M6P) and between the enzyme and the high-energy intermediate analogue inhibitor 5PAH are reported. Their analysis allows us to identify clearly the nature of each individual active site amino acid and to formulate a hypothesis for the overall mechanism of the reaction catalyzed by Type I PMIs, that is, the ring-opening and isomerization steps, respectively. Following enzyme-catalyzed ring-opening of β-M6P by zinc-coordinated water and Gln111 ligands, Lys136 is identified as the probable catalytic base involved in proton transfer between the two carbon atoms C1 and C2 of the substrate D-mannose 6-phosphate.
Laboratoire de Chimie Bioorganique et Bioinorganique, ICMMO, Univ Paris-Sud, UMR 8182, Orsay F-91405, France.
Céline
C
Roux
Forum
F
Bhatt
Johanna
J
Foret
Benoit
B
de Courcy
Nohad
N
Gresh
Jean-Philip
JP
Piquemal
Constance J
CJ
Jeffery
Laurent
L
Salmon
D000595:Amino Acid Sequence; D001667:Binding, Competitive; D002176:Candida albicans / Q000201:enzymology; D020134:Catalytic Domain; D004926:Escherichia coli / Q000201:enzymology; D005636:Fructosephosphates / Q000737:chemistry; D006801:Humans; D006834:Hydrazines / Q000737:chemistry; D006877:Hydroxamic Acids / Q000737:chemistry; D007700:Kinetics; D008359:Mannose-6-Phosphate Isomerase / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000737:chemistry*; D008360:Mannosephosphates / Q000737:chemistry; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D011487:Protein Conformation; D016415:Sequence Alignment; D013403:Sugar Phosphates / Q000737:chemistry
C558277:5-phospho-D-arabinonhydrazide; C456025:5-phosphoarabinonohydroxamic acid; D005636:Fructosephosphates; D006834:Hydrazines; D006877:Hydroxamic Acids; D008360:Mannosephosphates; D013403:Sugar Phosphates; C027693:mannose-6-phosphate; C027618:fructose-6-phosphate; D008359:Mannose-6-Phosphate Isomerase