An exploration of the binding site of aldolase using alkyl glycolamido phosphoric esters and alkyl monoglycolate phosphoric esters.
Alkyl glycolamido phosphoric esters (P-O-CH2-CO-NH-(CH2)n-CH3) and alkyl monoglycolate phosphoric esters (P-O-CH2-CO-O-(CH2)n-CH3), which are analogs of the aldolase substrate fructose-1-phosphate, were synthesized and use for probing the active site of rabbit muscle aldolase. The inhibition constants (Ki) were affected by the length of the alkyl groups of these compounds and a maximum value of Ki was observed between the number of methylene groups 2 and 4, depending on the type of compound. In the previous investigation, N-(omega-hydroxyalkyl)-glycolamido bisphosphoric esters (P-O-CH2-CO-NH-(CH2)n-O-P) and alkanediol monoglyclolate bisphosphoric esters (P-O-CH2-CO-O-(CH2)n-O-P) have a minimum Ki value between the number of methylene groups 1 and 4. The difference spectra of aldolase caused by binding of alkyl glycoamido phosphoric esters or alkyl monophosphates resembled that of their analogous bisphosphoric esters, but the intensity of absorbance was smaller than that of the bisphosphoric ester analogs. These results suggest that rabbit muscle aldolase has two binding sites for the phosphate groups on the entrance end of the active site cavity, the singly wound beta-barrel of the parallel alpha/beta class structure. The distance between the phosphate binding site Lys-107 in the beta-sheet structure (c) and Arg-148 in the beta-sheet structure (d) may possibly be expanded or contracted by the forms of the bending structure of the biphosphate compounds. Also, the change of distance between the beta-sheet structure (c) and (d) containing Trp-147, may have an effect on the environment of the tryptophan and cause a change of the absorbance of aldolase especially at 295-299 nm. On the other hand, the synthetic monophosphate compounds bind at only one of the two phosphate binding sites and have very little effect on the absorbance of Trp-147, in a similar manner as orthophosphate. The alkyl groups of monophosphate may be repelled by the ionic amino acid side chains, Asp-33, Lys-146, Glu-187 and/or Lys-229 in the middle of the active site cavity. However, the end of the long alkyl group of some monophosphates may possibly contact the hydrophobic bottom of the active site cavity without effect on the environment of Trp-147.
Ogata
H
H
Division of Chemistry and Biochemistry, The School of Allied Health Sciences, Yamaguchi University, Japan.
Fukuda
T
T
Yamamoto
K
K
Funakoshi
J
J
Takada
K
K
Yasue
N
N
Fujisaki
S
S
Kajigaeshi
S
S
D001665:Binding Sites; D001667:Binding, Competitive; D005634:Fructose-Bisphosphate Aldolase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D005636:Fructosephosphates / Q000378:metabolism*; D006016:Glycolates / Q000138:chemical synthesis* / Q000494:pharmacology; D009132:Muscles / Q000201:enzymology*; D010755:Organophosphates / Q000138:chemical synthesis* / Q000494:pharmacology
D005636:Fructosephosphates; D006016:Glycolates; D010755:Organophosphates; C032284:fructose-1-phosphate; C053956:glycolamide; D005634:Fructose-Bisphosphate Aldolase