The roles of the catalytic active-site residues aspartic acid-52 and glutamic acid-35 of chicken lysozyme (EC 3.2.1.17) have been investigated by separate in vitro mutagenesis of each residue to its corresponding amide (denoted as D52N and E35Q, respectively). The mutant enzyme D52N exhibits =5% of the wild-type lytic activity against Micrococcus luteus cell walls, while there is no measurable activity associated with E35Q (0.1% ± 0.1%). The measured dissociation constants for the chitotriose-enzyme complexes are 4.1 ,LM (D52N) and 13.4 pzM (E35Q) vs. 8.6 ,uM for wild type, indicating that the alterations in catalytic properties may be due in part to binding effects as well as to direct catalytic participation of these residues. The mutant lysozymes have been expressed in and secreted from yeast and obtained at a level of =a5 mg per liter of culture by high-salt elution from the cell walls.Chicken egg white lysozyme (CEWL; EC 3.2.1.17) has a distinguished history in the field of mechanistic enzymology. It was the first enzyme for which an atomic resolution x-ray structure was published (1), and the essence of the presently accepted catalytic mechanism was proposed almost exclusively from the structural information (2). The enzyme has additionally served as an important paradigm for studies in (i) the physical biochemistry of polydentate liganding, whereby the individual contributions to the several binding subsites have been separately evaluated (3); (ii) molecular evolution investigations (4, 5); and (iii) immunochemical studies (6). The enzyme is thus an attractive candidate for modification by site-directed mutagenesis, because the availability of specifically mutated constructs will have the potential to contribute significantly to all of the above investigations, as well as to studies on protein folding and stability. We report here the relatively high-efficiency expression of Strains. Plasmids were propagated in HB101 grown in LB medium (7). M13mpl8 subclones were grown in JM103 and in RZ1032 for mutagenesis (8). Saccharomyces cerevisiae strain GRF180 is leu2-3,-J12, ura3-52, his4-580, can (cir") and was derived from GRF18 by curing this strain of its endogenous 2-,um circle (9, 10). Escherichia coli and S. cerevisiae transformations were carried out as described (11,12).Plasmids. Plasmid pAB24 is a yeast-E. coli shuttle vector derived from pBR322 and pJDB219 (13). It contains the complete 2-gm circle and the LEU2-D and URA3 genes for selection and replication in yeast and pBR322 sequences and sequences for selection and replication in E. coli (14). Plasmid pAGAP1 is a derivative of pPGAP1 (15) that has the GAPDH-491 promoter replaced with a hybrid ADH2-GAPDH promoter fusion (14). Plasmid pLS1023 containing a CEWL cDNA clone (16) was the generous gift of G. Schutz (University of Cologne). DNA sequencing revealed two discrepancies between pLS1023 and the published sequence, which were repaired by site-directed mutagenesis.Site-Directed Mutagenesis of the Active Site. Two 21-base synthetic primers ...

FH is a tumor marker for bladder cancer. To reveal the presence of bladder cancer, the BTA TRAK assay detects FH, whereas FHR-1 is able to partly inhibit this detection. This indicates a special mechanism for a diagnostic immunoassay based on the combined effect of simultaneous positive and negative signals in a single sample.

While the elegance and efficiency of enzymatic catalysis have long tempted chemists and biochemists with reductionist leanings to try to mimic the functions of natural enzymes in much smaller peptides, such efforts have only rarely produced catalysts with biologically interesting properties. However, the advent of genetic engineering and hybridoma technology and the discovery ofcatalytic RNA have led to new and very promising alternative means of biocatalyst development. Synthetic chemists have also had some success in creating nonpeptide catalysts with certain enzyme-like characteristics, although their rates and specificities are generally much poorer than those exhibited by the best novel biocatalysts based on natural structures. A comparison of the various approaches from theoretical and practical viewpoints is presented. It is suggested that, given our current level of understanding, the most fruitful methods may incorporate both iterative selection strategies and rationally chosen small perturbations, superimposed on frameworks designed by nature.A thorough understanding of the chemical and structural bases of biological catalysis would lead to advances in medicine, synthetic chemistry, materials science, agriculture, and other fields. Such a level of insight, when it exists, will likely be signaled by a clear demonstration of the ability to construct, from first principles, a range of catalysts capable of transmuting both biopolymers and small molecules to desired products with high specificity and acceptable efficiency. The specialized proteins known as enzymes are the molecules that usually fill this role in nature, and the most likely means by which we could achieve the objective of catalysis to specification is by attaining an understanding of enzyme structure and function sufficient to allow design and construction of new molecules based on the same principles. This degree of understanding has been elusive, and there is as yet no case of an protein or peptide designed de novo that catalyzes a reaction of biological interest with efficiency and specificity comparable to that of natural enzymes or a novel reaction not carried out by enzymes. However, competing strategies of developing novel biocatalysts which make use of the frameworks of natural proteins have enjoyed considerable success in catalyzing both transformations analogous to the cognate reactions of natural enzymes and, in some cases, entirely novel reactions. The two major techniques in this category are catalytic antibodies (refs. 1 and 2; for a recent review, see ref.3) and reengineered natural enzymes (RNEs; for reviews, see refs. 4 and 5). Catalytic antibodies, which were originally reported in 1986, can be characterized as antibodies directed against haptens, which are usually synthetic analogs of the transition states of the chemical reactions to be catalyzed. Reengineering of enzymes has been possible in principle since the advent of genetic engineering, more than two decades ago; a review of Medline entries indicated that st...

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