A Double Mutation at the Tip of the Dimer Interface Loop of Triosephosphate Isomerase Generates Active Monomers with Reduced Stability
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- 作者：Wolfgang Schliebs ; Narmada Thanki ; Rainer Jaenicke ; and Rik K. Wierenga
- 刊名：Biochemistry
- 出版年：1997
- 出版时间：August 12, 1997
- 年：1997
- 卷：36
- 期：32
- 页码：9655 - 9662
- 全文大小：187K
- 年卷期：v.36,no.32(August 12, 1997)
- ISSN：1520-4995
文摘
Triosephosphate isomerase (TIM) is a very stable dimer. Inorder to understand better theimportance of dimerization for stability and catalytic activity, wehave constructed a monomeric double-mutation variant. The dimer interface residues Thr75 and Gly76,which are at the tip of loop 3, havebeen substituted by an arginine and a glutamate, respectively. Inwild type TIM, these two residues areat a distance of 27 Å from the active site (as measured within thesame subunit). This new variant,RE-TIM, was expressed in Escherichia coli, purified tohomogeneity, and biochemically characterized.Sedimentation equilibrium ultracentrifugation runs showed thatRE-TIM is a monomer in solution. Far-UV CD spectra indicate that this new variant is folded properly and thatthe secondary structure contentsof RE-TIM are similar to those of wild type TIM. The monomericRE-TIM has residual TIM activity.The thermal stability of RE-TIM is lower than that for wild typeTIM. CD melting curves for RE-TIMand wild type TIM show Tm values of 52 and 57
C, respectively, in the presence of the active siteligand2-phosphoglycolate at 1 mM. Previously, we have characterized twoother monomeric forms of TIM:monoTIM and H47N-TIM. The properties of RE-TIM, H47N-TIM, andmonoTIM are compared, and itis argued that the properties of RE-TIM will be very similar to thoseof wild type monomeric subunits.This implies that wild type monomeric subunits have some stabilityand are catalytically active. It is alsoinferred that these monomeric subunits have flexible loops whichrigidify at the dimer interface ondimerization, causing a 1000-fold increase ofkcat and a 10-fold decrease ofKm.
C, respectively, in the presence of the active siteligand2-phosphoglycolate at 1 mM. Previously, we have characterized twoother monomeric forms of TIM:monoTIM and H47N-TIM. The properties of RE-TIM, H47N-TIM, andmonoTIM are compared, and itis argued that the properties of RE-TIM will be very similar to thoseof wild type monomeric subunits.This implies that wild type monomeric subunits have some stabilityand are catalytically active. It is alsoinferred that these monomeric subunits have flexible loops whichrigidify at the dimer interface ondimerization, causing a 1000-fold increase ofkcat and a 10-fold decrease ofKm.