Almlract Blnnse, the extracellular dbonuclense of J~c~ inttrmedlm, b inhibited by banter, the natural protein inhibitor of the bontoloRons RNnse, barnnse, of R lntevmedius. The dissociation constants of the blnnse complexes with Imntar and Its double ~~la mutant are about 10"n M, only 5 to 43 times higher than those of the barnaes-bontar complex, As with bemuse, the denaturation temperature of blnnse is raised dramatically Ip the complex, Calorimetric studies of the formation and stability of the bin.se-bantar complex show that the blnase reaction with bamtar Is qualitatively similar to that of barnnse but some signif. kant quantitative differences are reported.

No abstract

The rate at which dinucleoside phosphates are cleaved by RNases is supposed to be determined by the mole fraction of enzyme-substrate complexes in which the phosphodiester moiety of a dinucleoside phosphate has a highly reactive conformation. The mole fraction of such complexes for a particular RNase depends on the nature of a nucleoside at the 05'-end of the phosphodiester bond. Experimental data are presented to support this hypothesis. RNaseStereoelectronic control specificity

Conformational stability of the aspartate aminotransferase has been studied. To this aim the isotherms of urea denaturation were obtained for the apoenzyme and the holoenzymes reconstituted with pyridoxal phosphate and its analogs. The state of the active site was shown to have an influence upon the general protein stability, the stability levels (the points of denaturation) were grouped around 5.1 M and 6.9 M urea depending on the analog structure.Proceeding from an idea of complementarity of free energy profiles in a congruent model system and in the corresponding protein moiety, the conformational stability was determined of the aspartate aminotransferase -inhibitor complexes simulating the enzyme -substrate ones which are formed a t the subsequent reaction steps. It was shown that a higher stablity of the protein corresponds to most of the high-energy complexes of the congruent system. It is suggested that in the case of aspartate aminotransferase the equalizing of the energy profile along the reaction coordinate is realized by means of few (perhaps two) discrete energy states of the protein globule.Redistributions of the electron density occurring during the chemical reactions catalysed by enzymes are coupled with conformational changes in the protein globulae. As protein does not radically change the chemical (covalent) nature of the intermediates but only affects their free energy levels both by the entropy and enthalpy factors [5]. A necessary condition for a reversible multistep process to be effective is the equalization of the free energy levels within the range of several kcal/mol. Hence, free energy of the protein globule must be changed in a complementary way in comparison with the congruent system to provide such an equalization. This argument is reviewed by Lamry and Biltonen [7].We are concerned with the experimental investigation of the changes in the stability of aspartate aminotransferase protein under different conditions. I n particular it was necessary to find out whether the protein macromolecule as a whole participated in the energy exchange or whether a relatively small part of the globule near the active site was involved in the process.Previous work with myoglobin, studying its resistance to denaturation factors [8], showed that binding of ligands to heme affected the conformational properties of the whole globule.This work concerns the isothermic unfolding of different forms of aspartate aminotransferase in aqueous-urea solutions : viz. apoenzyme, holoenzymc reconstructed with pyridoxal phosphate (pyridoxal-P), pyridoxamine phosphate (pyridoxamine-P), and Eur.

The work comprises : 1. Determination by nuclear magnetic resonance method of the conformation of the imino group of pyridoxal-P oxime in solution. It has been found to be nearly coplanar with the aromatic ring.2. Theoretical calculation by the method of atom-atom potentials of the permitted conformations of pyridoxal-P imine and its analogs : 6-methylpyridoxal-P and 5'-methylpyridoxal-P. There have been found seven favourable conformations for pyridoxal-P imine, some of which were prohibited in the analogs mentioned.3. Experimental estimation of the conformation of pyridoxal-Pimine in the active site of aspartate aminotransferase with the use of the above sterically hindered analogs and 5'-deoxy-5'-carboxymethylenepyridoxal analog. In result, the number of the possible conformations for pyridoxal-P imine in the active site has been reduced to a pair of symmetrical conformations.Pyridoxal5'-phosphate, the coenzyme of aspartate aminotransferase, is present in the active site of the enzyme in the form of an aldimine linked to the epsilon amino group of a lysine residue [l].The conformation of coenzyme moiety of the aldimine is specified by the following rotation angles around ordinary bonds: C, -C,, (x), C, -C,, (cp), 0 -C,, ($), and P -0 (0) (Fig. 1). An angle of 0" corresponds to a cis position of the bonds, adjoining the rotation axis. An increase in the magnitude of the angle corresponds to a clockwise rotation of a distant bond if one looks along the rotation axis. The cis positions are depicted in Fig. 1 .X-Ray analysis data of the crystals of pyridoxal-P and pyridoxamine-P derivatives [2 -61 have been recently published. The oxime of pyridoxal-P was shown to have a flat, competely extended conformation (1 = o", cp = 357" (-3"), $ = 174") [6]. In the derivatives lacking a double bond in position 4, the phosphate group may be present in one of several favourable conformations depending on the nature of the substituent in position 4 [2-61. The conformation of pyridoxal-P imines in solution was shown to depend on the ionization state of the polar groups of the molecule [7].Since pyridoxal-P is constrained by the apoenzyme by all its side groups [8], only one conformation of the molecule is present on the protein. Knowledge of this particular conformation is necessary prerequisite for understanding the real mechanism of enzymatic transamination.