Crystallographic analysis of trypsin-G226A. A specificity pocket mutant of rat trypsin with altered binding and catalysis.
The crystal structure of trypsin-G226A has been determined, in the presence of benzamidine, to a resolution of 1.75 A with an R-factor of 14.6%. The mutation was designed to alter substrate specificity by disrupting arginine binding, but was previously found to disrupt catalysis to a greater extent than binding. The arginine analog, benzamidine, has rotated 40 degrees and 49 degrees and translated 1.1 A in the specificity pocket, relative to the position in wild-type trypsin. The salt-bridge between the amidinium group of benzamidine and the carboxylate of D189 as well as four other hydrogen bonds have been replaced by a set of six new hydrogen bonds. Based on these interactions, computer modeling of an arginine substrate demonstrates that arginine terminal nitrogen atoms can occupy the new benzamidine nitrogen positions with torsion angle adjustments and without short contacts. In the secondary orientation, arginine substrates appear to be forced out of alignment with the active site. This may account for the larger drop in kcat with arginine relative to lysine substrates. A second possible cause of the altered activity is a change of the enzyme structure with concomitant loss of activity. No evidence of such a change is seen in the co-ordinates or temperature factors of the trypsin-G226A-benzamidine complex. A226 disrupts mainly the co-ordinates of amino acids with which it has direct contacts such that the effects of the mutation are absorbed locally.
Wilke
M E
ME
Department of Biochemistry and Biophysics, University of California, San Francisco 94143-0448.
Higaki
J N
JN
Craik
C S
CS
Fletterick
R J
RJ
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine; D001549:Benzamides / Q000494:pharmacology; D001665:Binding Sites; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D051381:Rats; D013379:Substrate Specificity; D014357:Trypsin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014961:X-Ray Diffraction / Q000379:methods
D001549:Benzamides; C037689:benzamide; D001120:Arginine; D014357:Trypsin