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Inosine monophosphate dehydrogenase (IMPDH) is an important target for immunosuppressive, antiviral and anticancer therapy. This enzyme catalyzes the key reaction in guanine nucleotide biosynthesis: the conversion of IMP into XMP with the concomitant reduction of NAD. The reaction involves a dehydrogenase step that produces NADH and a covalent E-XMP* intermediate and a hydrolysis step where E-XMP* is converted to XMP. We have solved the structure of the mizoribine monophosphate complex of IMPDH that resembles the transition state for the hydrolysis of E-XMP*. This structure reveals that IMPDH undergoes a large conformational change after NADH departs, transforming the enzyme into a hydrolase. This conformational change positions a mobile flap in the NADH site, with a conserved Arg-Tyr dyad adjacent to E-XMP*. Surprisingly, the Arg-Tyr dyad appears to act as the general base that activates water. The flap competes with drugs such as mycophenolic acid, so that the conformational change also appears to be a major determinant of drug selectivity.

The conversion of trypsin into a protease with chymotrypsin-like activity and specificity required substitution of fifteen residues in the S1 site and two surface loops with their chymotrypsin counterparts [Hedstrom,L., Szilagyi,L. and Rutter,W.J. (1992) Science, 255, 1249-1253]. These residues may define a set of general structural determinants of specificity in the trypsin family. In order to test this hypothesis, we have attempted to convert trypsin into a protease with specificity for substrates containing small aliphatic residues by replacing the S1 site and these surface loops with the analogous residues of elastase. Five elastase-like mutant enzymes were constructed with various combinations of these substitutions. Four mutant enzymes catalyze the hydrolysis of MeOSuc-Ala-Ala-Pro-Ala-SBzl more efficiently than the hydrolysis of Suc-Ala-Ala-Pro-Phe-SBzl. This observation indicates that the mutant enzymes have elastase-like esterase specificity. The best mutant, Tr-->E1-2, is a more specific esterase than elastase: the ratio of the values of kcat/Km for MeOSuc-Ala-Ala-Pro-Ala-SBzl and Suc-Ala-Ala-Pro-Phe-SBzl is greater than 160 for Tr-->E1-2 and 50 for elastase. However, the esterase activity of Tr-->E1-2 is 300-fold less than elastase; in addition, Tr-->E1-2 has no measurable amidase activity. Thus these substitutions do not construct a protease with elastase-like activity. These experiments indicate that a unique structural solution is required for each different specificity. Previous work suggested that instability of the S1 site is a major barrier to redesigning the specificity of trypsin. This view is corroborated by preliminary structural studies of Tr-->E1-2. One dimensional 1H NMR spectrum of Tr-->E1-2 suggests that the S1 site and the two surface loops of this mutant trypsin may be disordered.

The S1 binding site of trypsin is cross-linked by the conserved Cys191-Cys220 disulfide bond. The substitution of Cys191 and Cys220 with Ala decreases the activity of trypsin by 20-200-fold as measured by kcat/K(m) for the hydrolysis of amide substrates; in contrast, ester hydrolysis is decreased by < 10-fold. Similar decreases are observed in the hydrolysis of oligopeptide and single amino acid substrates. This decrease in activity results from a decrease in the acylation rate. The substrate binding and deacylation rate are not affected by the loss of the disulfide bond. C191A/C220A binds BPTI with the same affinity as trypsin, although the affinity of benzamidine is decreased 10-fold and the affinity of leupeptin is decreased 1000-fold. The CD spectrum of C191A/C220A displays significant differences from that of trypsin; these differences most likely result from the loss of the disulfide chromophore, although perturbation of enzyme structure cannot be discounted. The loss of the Cys191-Cys220 disulfide has no effect on the stability of trypsin as measured by urea denaturation. Single and double substitutions of Ser at positions 191 and 220 have a similar activity to C191A/C220A. These results indicate that the Cys191-Cys220 disulfide bond is not essential for the function, structure or stability of trypsin.

We describe a simple and effective procedure to screen for active proteases among a large number of mutants. First, the mutants are genetically tested by the protease activity produced in the periplasm of transformed bacteria which supplies the cells with a nitrogen source by hydrolyzing a protein applied to plates. Then a less sensitive activity staining and an X-ray film digestion assay are used to verify and estimate the activity of the mutants that proved to be positive in the first step. Depending essentially on the level of periplasmic protease activity, the method can detect both the activity and the stability of the expressed enzymes. We calibrated the method with transformants that produce wild-type trypsin, chymotrypsin and trypsin mutants of known activity. Using this method we found two active revertants of the inactive Asn102 trypsin mutant, by screening approximately 4.4 x 10(4) random mutants that were generated by the polymerase chain reaction on a cDNA fragment. This procedure should be useful in searching for proteases of novel specificity and/or reaction chemistry engineered by random mutagenesis, and also for in vitro evolution studies.