PubMed · 4308632
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T Kitagawa. 1969. [Glycogen metabolism abnormalities].. https://pubmed.ncbi.nlm.nih.gov/4308632/
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The interactions of a transition state mimic anilinomethyl glucoimidazole (AmGlcIm), with a K(i) constant of 0.6 x 10(-)(9) M and a Gibbs free energy value of -53.5 kJ/mol, with a family GH3 beta-d-glucan glucohydrolase from barley have been analyzed crystallographically and by ab initio quantum mechanical modeling. AmGlcIm binds 3 times more tightly to the beta-d-glucan glucohydrolase than a previously investigated phenyl glucoimidazole. In the enzyme-AmGlcIm complex, an additional residue, Tyr253, and a water molecule positioned between subsites -1 and +1 are recruited for binding. Analyses of the two binary complexes reveal the following. (i) An intricate network exists in which hydrogen bonds between the enzyme's catalytic pocket residues Lys206, His207, Tyr253, Asp285, and Glu491 and the glucoimidazoles are shorter by 0.15-0.53 A, compared with distances of hydrogen bonds in the Michaelis complex. (ii) The "glucose" moiety of the glucoimidazoles adopts a (4)E conformation that is vital for the low-nanomolar binding. (iii) The N1 atoms of the glucoimidazoles are positioned nearly optimally for in-line protonation by the Oepsilon1 atom of the catalytic acid/base Glu491. (iv) The enzyme derives binding energies from both glycone and aglycone components of the glucoimidazoles. (iv) The prevalent libration motion of the two domains of the enzyme could play a significant role during induced fit closure in the active site. (v) Modeling based on the structural data predicts that protons could be positioned on the N1 atoms of the glucoimidazoles, and the catalytic acid/base Glu491 could carry an overall negative charge. (vi) The enzyme-AmGlcIm complex reveals the likely structure of an early transition state during hydrolysis. Finally, the high-resolution structures enabled us to define minimal structures of oligosaccharides attached to Asn221, Asn498, and Asn600 N-glycosylation sites.
Among the numerous well-characterized families of glycosidases, family 4 appears to be the anomaly, requiring both catalytic NAD+ and a divalent metal for activity. The unusual cofactor requirement prompted the proposal of a mechanism involving key NAD+-mediated redox steps as well as elimination of the glycosidic oxygen. Primary kinetic isotope effects for the 2- and 3-deutero substrate analogues, isotopic exchange with solvent, and structural analysis of a 6-phospho-beta-glucosidase, BglT (E.C. 3.2.1.6), provided evidence in support of the proposed mechanism, which has striking resemblances to that of the sugar dehydratases. Furthermore, analysis of the stereochemical outcome indicated that family 4 enzymes are retaining glycosidases.
When roots of 5-d-old red clover (Trifolium pratense L.) seedlings were treated with chitohexaose and CuCl(2), constitutive glucosidic conjugates of formononetin (F) and (-)-maackiain (Ma) promptly disappeared. Free F and Ma, which were not detected in the control tissues, rapidly appeared to reach the maximum levels 24 h after the initiation of treatment and then declined. The pattern of appearance and disappearance was the same between the tissues treated with chitohexaose and CuCl(2). The enzyme activities related to isoflavonoid metabolism were investigated using crude extracts from elicitor-treated roots. The conjugate-forming glucosyltransferase and malonyltransferase activities were lost or markedly reduced after elicitor treatment. On the other hand, malonylesterase and glucosidase activities remained unchanged or showed only slight increase. Phenylalanine ammonia-lyase activity disappeared following elicitor treatment. These results indicated that free aglycones were produced from the conjugate pool by hydrolysis under conditions in which the biosynthetic pathway was extinguished. The amount of Ma produced did not explain that of MaGM lost (about 45%). Since Ma, but not its conjugates, served as a substrate for peroxidase from the elicitor-treated roots, Ma was considered to be converted to insoluble materials.