Catalytic properties and conformation of hydrophobized alpha-chymotrypsin incorporated into a bilayer lipid membrane.
A set of artificially hydrophobized alpha-chymotrypsin derivatives, carrying 2-11 stearoyl residues per enzyme molecule, were synthesized and their catalytic parameters and conformation in water solution and in the liposome-bound state were investigated. Hydrophobization of alpha-chymotrypsin and its further incorporation into phosphatidylcholine (PC) liposomes have no effect on the rate constant of the N-acetyl-L-tyrosine ethyl ester (ATEE) ester bond hydrolysis (k(cat)). At the same time, an increase in the number of stearoyl residues attached to the enzyme results in a drastic decrease of ATEE binding to the active center (K(M) increase). Incorporation of the hydrophobized enzyme into the PC liposome membrane results in K(M) recovery to nearly that of native alpha-chymotrypsin. The above changes are accompanied by partial unfolding of the enzyme molecules observed by fluorescence measurements. The obtained results are of interest to mimic the contribution of surface hydrophobic sites in the functioning of membrane proteins.
Kozlova
N O
NO
Department of Polymer Science, School of Chemistry, Moscow State University, Leninskiye Gory, Moscow, Russian Federation.
Bruskovskaya
I B
IB
Melik-Nubarov
N S
NS
Yaroslavov
A A
AA
Kabanov
V A
VA
D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002918:Chymotrypsin / Q000737:chemistry* / Q000378:metabolism; D006868:Hydrolysis; D008051:Lipid Bilayers / Q000737:chemistry*; D008081:Liposomes; D010713:Phosphatidylcholines; D011485:Protein Binding; D011487:Protein Conformation; D011510:Proteolipids / Q000737:chemistry*; D013229:Stearic Acids / Q000737:chemistry; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism
D008051:Lipid Bilayers; D008081:Liposomes; D010713:Phosphatidylcholines; D011510:Proteolipids; D013229:Stearic Acids; C020485:proteoliposomes; D014443:Tyrosine; C007651:ethyl N-alpha-acetyl-tyrosinate; D002918:Chymotrypsin; C117608:alpha-chymotrypsin