COOPERATIVITY OF PAPAIN SUBSTRATE INTERACTION ENERGIES IN THE S2 TO S2' SUBSITES

被引:53
作者
BERTI, PJ
FAERMAN, CH
STORER, AC
机构
[1] BIOTECHNOL RES INST,PROT ENGN SECT,6100 AVE ROYALMOUNT,MONTREAL H4P 2R2,QUEBEC,CANADA
[2] MCGILL UNIV,DEPT BIOCHEM,MONTREAL H3G 1Y6,QUEBEC,CANADA
关键词
D O I
10.1021/bi00219a033
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Enzyme-substrate contacts in the hydrolysis of ester substrates by the cysteine protease papain were investigated by systematically altering backbone hydrogen-bonding and side-chain hydrophobic contacts in the substrate and determining each substrate's kinetic constants. The observed specificity energies [defined as DELTA-DELTA-G(obs) = -RT ln [K(cat)/K(M)) first/(K(cat)/K(M)second)]] of the substrate backbone hydrogen bonds were -2.7 kcal/mol for the P2 NH and -2.6 kcal/mol for the P1 NH when compared against substrates containing esters at those sites. The observed binding energies were -4.0 kcal/mol for the P2 Phe side chain, -1.0 kcal/mol for the P1' C=O, and -2.3 kcal/mol for the P2' NH. The latter three values probably all significantly underestimate the incremental binding energies. The P2 NH, P2 Phe side-chain, and P1 NH contacts display a strong interdependence, or cooperativity, of interaction energies that is characteristic of enzyme-substrate interactions. This interdependence arises largely from the entropic cost of forming the enzyme-substrate transition state. As favorable contacts are added successively to a substrate, the entropic penalty associated with each decreases and the free energy expressed approaches the incremental interaction energy. This is the first report of a graded cooperative effect. Elucidation of favorable enzyme-substrate contacts remote from the catalytic site will assist in the design of highly specific cysteine protease inhibitors.
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页码:1394 / 1402
页数:9
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