The kinetics of the trypsin with several esters derived from p-amidinophenol have been studied. These esters are characterized by their linkage, i.e. the site-specific group for the enzyme. (chaeged amidinium) is not involved in their carbonyl portion but in the leaving group. These esters were demonstrated to undergo efficient and specific tryptic hydrolysis. The selectivity and efficiency of p-amidinophenyl esters were compared to those of normal-type substrates, and it was concluded that these esters are almost comparable to normal-type substrates both in binding and acylation. Thus, new term, "inverse substrates" was proposed for these esters. This has provided, for the first time, a general method for the specific introduction of an acyl group carrying a non-specific residue into the trypsin active site without recourse to a cationic acyl moiety characteristic of conventional substrates. Acyl trypsins containing "reporter group" such as fluorophore, stable free radical and optically active chromophore were prepared in a very specific manner by use of "inverse substrates". The properties of acyl trypsins were spectrometrically analyzed with a view to elucidating the microenvironment of the active site. Deacylation rates of acyl trypsins carrying D-amino acid residues were determined for the first time. The steric requirements of active site of trypsin were analyzed. The effects of positively charged ligands on the deacylaction process were also studied in connection with the requirements of the active site.
The chemical substitution of ferrous protoporphyrin IX with cobaltous protoporphyrin IX in myoglobin (Mb) and hemoglobin (Hb) has allowed for the detailed investigation of modes of interaction among molecular oxygen, the prothetic group, and the apoprotein moiety in these oxygen carrying hemoproteins. We have compared the ligand orientation of oxy meso-CoMb with that of oxy proto-CoMb by single crystal EPR spectroscopy at ambient and cryogenic temperatures. Single orientation was found at ambient temperature, but upon freezing two unequivalent orientations of O-O axis were found in both cobalt myoglobin single crystals. On the otherhand, two different ν(O-O) stretching vibrations were observed in oxy CoMb by resonance Raman spectroscopy at ambient temperature. We have demonstrated that metal-ligand geometry in these Co (II) complexes in unfrozen fluid and frozen solid states is altered by chemical modification at 2, 4-porphyrin peripheral groups.