Generation of a large combinatorial library of the immunoglobulin repertoire in phage lambda

A novel bacteriophage lambda vector system was used to express in Escherichia coli a combinatorial library of Fab fragments of the mouse antibody repertoire. The system allows rapid and easy identification of monoclonal Fab fragments in a form suitable for genetic manipulation. It was possible to generate, in 2 weeks, large numbers of monoclonal Fab fragments against a transition state analog hapten. The methods described may supersede present-day hybridoma technology and facilitate the production of catalytic and other antibodies.     

Catalytic antibodies with lipase activity and R or S substrate selectivity

The specific hydrolysis of unactivated esters bearing an R or S enantiomeric alcohol has been achieved by two separate classes of catalytic antibodies induced to bind either the R or S substrates. The antibodies exhibit rate accelerations (10(3) to 10(5] above background hydrolysis that, coupled with their antipodal specificity, provide a novel set of reagents for use in synthesis.     

Reversible compartmentalization of de novo purine biosynthetic complexes in living cells

Purines are synthesized de novo in 10 chemical steps that are catalyzed by six enzymes in eukaryotes. Studies in vitro have provided little evidence of anticipated protein-protein interactions that would enable substrate channeling and regulation of the metabolic flux. We applied fluorescence microscopy to HeLa cells and discovered that all six enzymes colocalize to form clusters in the cellular cytoplasm. The association and dissociation of these enzyme clusters can be regulated dynamically, by either changing the purine levels of or adding exogenous agents to the culture media. Collectively, the data provide strong evidence for the formation of a multi-enzyme complex, the "purinosome," to carry out de novo purine biosynthesis in cells.     

The enzymic nature of antibody catalysis: development of multistep kinetic processing

Detailed kinetic investigations of a catalytic antibody that promotes the hydrolyses of an anilide and phenyl ester show that this catalyst uses a multistep kinetic sequence resembling that found in serine proteases to hydrolyze its substrates, although antibody was elicited to a single transition-state analog. Like the serine proteases the antibody catalyzes the hydrolysis reactions through a putative covalent intermediate, but unlike the enzymes it may use hydroxide ion to cleave the intermediates. Nevertheless, the antibody is a potent catalyst with turnover at higher pH values rivaling that of chymotrypsin. This analysis also reveals that turnover by the antibody is ultimately limited by product desorption, suggesting that improvements in catalytic efficiency may be achieved by judicious changes in the structure of the substrate, so that it is not superimposable on that of the eliciting hapten.     

An antifungal agent inhibits an aminoacyl-tRNA synthetase by trapping tRNA in the editing site

Aminoacyl-transfer RNA (tRNA) synthetases, which catalyze the attachment of the correct amino acid to its corresponding tRNA during translation of the genetic code, are proven antimicrobial drug targets. We show that the broad-spectrum antifungal 5-fluoro-1,3-dihydro-1-hydroxy-2,1-benzoxaborole (AN2690), in development for the treatment of onychomycosis, inhibits yeast cytoplasmic leucyl-tRNA synthetase by formation of a stable tRNA(Leu)-AN2690 adduct in the editing site of the enzyme. Adduct formation is mediated through the boron atom of AN2690 and the 2'- and 3'-oxygen atoms of tRNA's3'-terminal adenosine. The trapping of enzyme-bound tRNA(Leu) in the editing site prevents catalytic turnover, thus inhibiting synthesis of leucyl-tRNA(Leu) and consequentially blocking protein synthesis. This result establishes the editing site as a bona fide target for aminoacyl-tRNA synthetase inhibitors.     

At the crossroads of chemistry and immunology: catalytic antibodies

Immunochemistry has historically focused on the nature of antigenicity and antibody-antigen recognition. However, in the last 5 years, the field of immunochemistry has taken a new direction. With the aid of mechanistic and synthetic chemistry, the vast network of molecules and cells of the immune system has been tapped to produce antibodies with a new function--catalytic antibodies. Because antibodies can be generated that selectively bind almost any molecule of interest, this new technology offers the potential to tailor-make highly selective catalysts for applications in biology, chemistry, and medicine. In addition, catalytic antibodies provide fundamental insight into important aspects of biological catalysis, including the importance of transition-state stabilization, proximity effects, general acid and base catalysts, electrophilic and nucleophilic catalysis, and strain.     

U.S. Sulfur Dioxide Emissions Trading Program: Results and Further Applications

The use of emissions trading (cap and trade) is gaining worldwide recognition as an extremely effective policy tool. More than 4 million tons of SO₂ have been reduced annually from sources participating in the US SO₂ emissions trading program. Ambient SO₂ levels and sulfate deposition have decreased by over 25 percent in the most sensitive ecosystems. By harnessing market forces, the program has led to significant cost savings. This paper discusses the results of the US SO₂ emissions trading program and emerging efforts in the US and around the world to use emissions trading.     

A dynamic knockout reveals that conformational fluctuations influence the chemical step of enzyme catalysis

Conformational dynamics play a key role in enzyme catalysis. Although protein motions have clear implications for ligand flux, a role for dynamics in the chemical step of enzyme catalysis has not been clearly established. We generated a mutant of Escherichia coli dihydrofolate reductase that abrogates millisecond-time-scale fluctuations in the enzyme active site without perturbing its structural and electrostatic preorganization. This dynamic knockout severely impairs hydride transfer. Thus, we have found a link between conformational fluctuations on the millisecond time scale and the chemical step of an enzymatic reaction, with broad implications for our understanding of enzyme mechanisms and for design of novel protein catalysts.     

A perspective on enzyme catalysis

The seminal hypotheses proposed over the years for enzymatic catalysis are scrutinized. The historical record is explored from both biochemical and theoretical perspectives. Particular attention is given to the impact of molecular motions within the protein on the enzyme's catalytic properties. A case study for the enzyme dihydrofolate reductase provides evidence for coupled networks of predominantly conserved residues that influence the protein structure and motion. Such coupled networks have important implications for the origin and evolution of enzymes, as well as for protein engineering.