TY - JOUR
T1 - Structure of a Ricin Mutant Showing Rescue of Activity by a Noncatalytic Residue
AU - Kim, Youngsoo
AU - Mlsna, Debra
AU - Monzingo, Arthur F.
AU - Ready, Michael P.
AU - Frankel, Art
AU - Robertus, Jon D.
PY - 1992
Y1 - 1992
N2 - Ricin A chain is an N-glycosidase which removes a single adenine base from a conservative loop of 28S rRNA, thereby inactivating eukaryotic ribosomes. The mechanism of action has been proposed to include transition-state stabilization of an oxycarbonium ion on the substrate ribose by interaction with Glu 177. Conversion of Glu 177 to Gin reduces activity nearly 200-fold [Ready, M. P., Kim, Y., & Robertus, J. D. (1991) Proteins. Struct., Funct., Genet. 10, 270-278] while conversion to Ala (E177A) reduces activity only 20-fold [Schlossman, D., Withers, D., Welsh, P., Alexander, A., Robertus, J., & Frankel, A. (1989) Mol. Cell. Biol. 9, 5012-5021]. X-ray analysis of the latter mutant protein shows that a residue at the edge of the active site, Glu 208, rotates into the space left vacant by the mutation. Its rearranged carboxylate partially substitutes for that of Glu 177. This is equivalent to the rescue of enzyme activity by a second-site reversion. Kinetic analysis shows the E177A mutation affects kcat and not Km, consistent with the notion that the carboxylate serves in transition-state stabilization.
AB - Ricin A chain is an N-glycosidase which removes a single adenine base from a conservative loop of 28S rRNA, thereby inactivating eukaryotic ribosomes. The mechanism of action has been proposed to include transition-state stabilization of an oxycarbonium ion on the substrate ribose by interaction with Glu 177. Conversion of Glu 177 to Gin reduces activity nearly 200-fold [Ready, M. P., Kim, Y., & Robertus, J. D. (1991) Proteins. Struct., Funct., Genet. 10, 270-278] while conversion to Ala (E177A) reduces activity only 20-fold [Schlossman, D., Withers, D., Welsh, P., Alexander, A., Robertus, J., & Frankel, A. (1989) Mol. Cell. Biol. 9, 5012-5021]. X-ray analysis of the latter mutant protein shows that a residue at the edge of the active site, Glu 208, rotates into the space left vacant by the mutation. Its rearranged carboxylate partially substitutes for that of Glu 177. This is equivalent to the rescue of enzyme activity by a second-site reversion. Kinetic analysis shows the E177A mutation affects kcat and not Km, consistent with the notion that the carboxylate serves in transition-state stabilization.
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U2 - 10.1021/bi00127a035
DO - 10.1021/bi00127a035
M3 - Article
C2 - 1348187
AN - SCOPUS:0026740874
SN - 0006-2960
VL - 31
SP - 3294
EP - 3296
JO - Biochemistry
JF - Biochemistry
IS - 12
ER -