NMR-STUDIES OF INTERNAL DYNAMICS OF SERINE PROTEINASE PROTEIN INHIBITORS - BINDING REGION MOBILITIES OF INTACT AND REACTIVE-SITE HYDROLYZEDCUCURBITA-MAXIMA TRYPSIN-INHIBITOR (CMTI)-III OF THE SQUASH FAMILY AND COMPARISON WITH THOSE OF COUNTERPARTS OF CMTI-V OF THE POTATO-I FAMILY

Serine proteinase protein inhibitors follow the standard mechanism of inhibition (Laskowski M Jr, Kato I, 1980, Annu Rev Biochem 49:593-626) , whereby an enzyme-catalyzed equilibrium between intact (I) and react ive-site hydrolyzed inhibitor (I) is reached. The hydrolysis constant , K-hyd, is defined as [I]/[I]. Here, we explore the role of internal dynamics in the resynthesis of the scissile bond by comparing the int ernal mobility data of intact and cleaved inhibitors belonging to two different families. The inhibitors studied are recombinant Cucurbita m axima trypsin inhibitor III (rCMTI-III; M-r 3 kDa) of the squash famil y and rCMTI-V (M-r similar to 7 kDa) of the potato I family. These two inhibitors have different binding loop-scaffold interactions and diff erent K-hyd values-2.4 (CMTI-III) and 9 (CMTI-V)-at 25 degrees C. The reactive-site peptide bond (P-1-P-1') is that between Arg(5) and Ile(6 ) in CMTI-III, and that between Lys(44) and Asp(45) in CMTI-V. The ord er parameters (S-2) of backbone NHs of uniformly N-15-labeled rCMTI-II I and rCMTI-III were determined from measurements of N-15 spin-lattic e and spin-spin relaxation rates, and {H-1}-N-15 steady-state heteronu clear Overhauser effects, using the model-free formalism, and compared with the data reported previously for rCMTI-V and rCMTI-V. The backb ones of rCMTI-III ([S-2] = 0.71) and rCMTI-III ([S-2] = 0.63) are mor e flexible than those of rCMTI-V ([S-2] = 0.83) and rCMTI-V ([S-2] = 0.85). The binding loop residues, P-4-P-1, in the two proteins show th e following average order parameters: 0.57 (rCMTI-III) and 0.44 (rCMTI -III); 0.70 (rCMTI-V) and 0.40 (rCMTI-V*). The P-1'-P-4' residues, on the other hand, are associated with [S-2] values of 0.56 (rCMTI-III) and 0.47 (rCMTI-III); and 0.73 (rCMTI-V) and 0.83 (rCMTI-V*). The new ly formed C-terminal (P-n residues) gains a smaller magnitude of flexi bility in rCMTI-III due to the Cys(3)-Cys(20) crosslink. In contrast, the newly formed N-terminal (P-n' residues) becomes more flexible onl y in rCMTI-III, most likely due to lack of an interaction between the P-1' residue and the scaffold in rCMTI-III. Thus, diminished flexibil ity gain of the P-n residues and, surprisingly, increased flexibility of the P-n' residues seem to facilitate the resynthesis of the P-1-P-1 ' bond, leading to a lower K-hyd value.