Expression of Functionality of dlhymotrypsin AN ALTERNATE BINDING MODE IN THE SUBSTRATE SPECIFICITY SITE*

Three-dimensional 2.8 A resolution x-ray crystallographic studies show that toluenesulfonamide and pipsylamide bind isomorphously in the aromatic specificity binding site of (Y-chymotrypsin. However, their orientation differs by about 90” from that usually associated with substrate-like molecules, suggesting a nonproductive binding mode. A second- ary binding site is also operative in one molecule of the dimer of the pipsylamide derivative and it is located some 22 w from the active site; however, this site is not operative in the toluenesulfonamide derivative. Binding of toluenesulfonamide and pipsylamide in the specificity site occurs without inducing any significant changes in the native enzyme structure, in contrast to the behavior observed upon tosylation or upon transition state analogue binding of phenylethaneboronic acid. The structural changes accompanying the formation of the latter derivatives are generally asymmetric with respect to the dimeric structure of CX-chymotrypsin and are generally confined to the binding domain or cylinder 2 of the enzyme (sequence >122). These changes are displayed in a new way via diagonal distance map representation.

Three-dimensional 2.8 A resolution x-ray crystallographic studies show that toluenesulfonamide and pipsylamide bind isomorphously in the aromatic specificity binding site of (Ychymotrypsin. However, their orientation differs by about 90" from that usually associated with substrate-like molecules, suggesting a nonproductive binding mode. A secondary binding site is also operative in one molecule of the dimer of the pipsylamide derivative and it is located some 22 w from the active site; however, this site is not operative in the toluenesulfonamide derivative. Binding of toluenesulfonamide and pipsylamide in the specificity site occurs without inducing any significant changes in the native enzyme structure, in contrast to the behavior observed upon tosylation or upon transition state analogue binding of phenylethaneboronic acid. The structural changes accompanying the formation of the latter derivatives are generally asymmetric with respect to the dimeric structure of CXchymotrypsin and are generally confined to the binding domain or cylinder 2 of the enzyme (sequence >122). These changes are displayed in a new way via diagonal distance map representation.  Table I, from which it can be seen that they are quite isomorphous with those of u-chymotrypsin.
Three-dimensional sets of 2.8 A resolution intensity data were collected from these crystals and converted to "best" difference electron density maps in a manner similar to that described elsewhere (3,4).   The space group and unit cell dimensions were in error, owing in part to the extremely small size of the crystals then The precession photograph (Fig. 2) of the hk0 zone based on the incorrect choice of axes therefore corresponds ta the HKH zone based on the correct choice of axes.

AND DISCUSSION
By this transformation, the correct orthorhombic cell parameters can be calculated as A = 52.37, B = 43.23, and C = 28.72 A which are in close agreement with the newly observed values. The unit cell volume for the orthorhombic cell is thrice that for the monoclinic cell.
The revised VM value is 2.39 A3/dalton on the basis of the assumption that the asymmetric unit contains 1 molecule. The solvent content of the crystal was re-estimated to be 51% by volume. The diffraction data extend to well beyond 2 A spacings.
Three heavy atom derivatives have been obtained with the use of uranium, platinum, and gold reagents, and x-ray data sets have been collected to 2.8 A for the native crystals and the uranium derivative, and to 5.6 A for the platinum and gold derivatives. The determination of the three-dimensional struc-Line 7 should read "I Ap [ > 0.12 eAT3" instead of "Ap > 0.12 eAb3. The sentence "Activity and pH determination was performed immediately." should be changed as follows: pH determination was performed immediately, whereas activities were measured after dialysis of each fraction against ture is currently under way in our laboratory. 0.02 M sodium phosphate buffer, bH 7.1.
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