The Interaction of Fusarium solani pisi Cutinase with Long Chain Spin Label Esters

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• 作者：Kristian R. Poulsen ; Thomas K. S//pubs.acs.org/images/gifchars/oslash.gif" border="0">rensen ; Laurent Duroux ; Evamaria I. Petersen ; Steffen B. Petersen ; Reinhard Wimmer
• 刊名：Biochemistry
• 出版年：2006
• 出版时间：August 1, 2006
• 年：2006
• 卷：45
• 期：30
• 页码：9163 - 9171
• 全文大小：680K
• 年卷期：v.45,no.30(August 1, 2006)
• ISSN：1520-4995

文摘

We here present a study of the interaction between the Fusarium solani pisi cutinase mutantS120A and spin-labeled 4,4-dimethyloxazoline-N-oxyl-(DOXYL)-stearoyl-glycerol substrates in a micellarsystem. The interaction is detected by NMR measuring changes in chemical shift for ¹H and ¹⁵N as wellas relaxation parameters for backbone ¹H (T₁) and ¹⁵N (T₁, T₂) atoms as well as for side chain methylgroups ¹H (T₁). The detected interaction shows a weak binding of cutinase to the lipid micelles. Structuraland mobility changes are located inside and around the active site, its flanking loops, and the oxyanionhole, respectively. Relaxation changes in the amino acid pairs Ser 92, Ala 93 and Thr 173, Gly 174positioned at the edge of each of the active site flanking loops make these residues prime candidates forhinges, allowing for structural rearrangement during substrate binding. The cutinase mutant S120A usedcarries a 15 amino acid pro-peptide; the significance of this pro-peptide was so far undetermined. Weshow here that the pro-peptide is affected by the presence of the micellar substrate. Relaxation enhancementsindicative of spatial proximity between the DOXYL group in the lipid chain and some hydrophobic residuessurrounding the active site could be found.