Protein-Free 50S Ribosomal Subunits Catalyze

Peptide Bond Formation In Vitro

Perhaps the most significant case of catalysis by RNA occurs in protein synthesis. Harry F. Noller and his colleagues have found that the peptidyl transferase reaction, which is the reaction of peptide bond formation during protein synthesis (Figure 14.24), can be catalyzed by 50S ribosomal subunits (see Chapter 12) from which virtually all of the protein has been removed. These experiments imply that just the 23S rRNA by itself is capable of catalyzing peptide bond formation. Also, the laboratory of Thomas Cech has created a synthetic 196-nucleotide-long ribozyme capable of performing the peptidyl transferase reaction.

Figure 14.24 •Protein-free 50S ribosomal subunits have peptidyl transferase activity. Peptidyl transferase is the name of the enzymatic function that catalyzes peptide bond formation. The presence of this activity in protein-free 50S ribosomal subunits was demonstrated using a model assay for peptide bond formation in which an aminoacyl-tRNA analog (a short RNA oligonucleotide of sequence CAACCA carrying³⁵ S-labeled methionine attached at its 3'-OH end) served as the peptidyl donor and puromycin (another amino-acyl-tRNA analog) served as the peptidyl acceptor. Activity was measured by monitoring the formation of ³⁵ S-labeled methioninyl-puromycin.

Several features of these “RNA enzymes,” or ribozymes, lead to the realization that their biological efficiency does not challenge that achieved by proteins. First, RNA enzymes often do not fulfill the criterion of catalysis in vivo because they act only once in intramolecular events such as self-splicing. Second, the catalytic rates achieved by RNA enzymes in vivo and in vitro are significantly enhanced by the participation of protein subunits. Nevertheless, the fact that RNA can catalyze certain reactions is experimental support for the idea that a primordial world dominated by RNA molecules existed before the evolution of DNA and proteins.

Catalytic Antibodies: Abzymes

Antibodies are immunoglobulins, which, of course, are proteins. Like other antibodies, catalytic antibodies, so-called abzymes, are elicited in an organism in response to immunological challenge by a foreign molecule called an antigen (see Chapter 29 for discussions on the molecular basis of immunology). In this case, however, the antigen is purposefully engineered to be an analog of the transition-state intermediate in a reaction. The rationale is that a protein specific for binding the transition-state intermediate of a reaction will promote entry of the normal reactant into the reactive, transition-state conformation. Thus, a catalytic antibody facilitates, or catalyzes, a reaction by forcing the conformation of its substrate in the direction of its transition state. (A prominent explanation for the remarkable catalytic power of conventional enzymes is their great affinity for the transition-state intermediates in the reactions they catalyze; see Chapter 16.)

Figure 14.25 •Catalytic antibodies are designed to specifically bind the transition-state intermediate in a chemical reaction. (a) The intramolecular hydrolysis of a hydroxy ester to yield as products a d-lactone and the alcohol phenol. Note the cyclic transition state. (b) The cyclic phosphonate ester analog of the cyclic transition state. Antibodies raised against this phosphonate ester act as enzymes: they are catalysts that markedly accelerate the rate of ester hydrolysis.

One strategy has been to prepare ester analogs by substituting a phosphorus atom for the carbon in the ester group (Figure 14.25). The phospho-compound mimics the natural transition state of ester hydrolysis, and antibodies elicited against these analogs act like enzymes in accelerating the rate of ester hydrolysis as much as 1000-fold. Abzymes have been developed for a number of other classes of reactions, including COC bond formation via aldol condensation (the reverse of the aldolase reaction [see Figure 14.2, reaction 4 and Chapter 19]) and the pyridoxal 5'-P-dependent aminotransferase reaction shown in Figure 14.22. In this latter instance, N_a-(5'-phosphopyridoxyl)-lysine (Figure 14.26a) coupled to a carrier protein served as the antigen. An antibody raised against this antigen catalyzed the conversion of d-alanine and pyridoxal 5'-P to pyruvate and pyridoxamine 5'-P (Figure 14.26b). This biotechnology offers the real possibility of creating “designer enzymes,” specially tailored enzymes designed to carry out specific catalytic processes.

Figure 14.26 •(a) Antigen used to create an abzyme with aminotransferase activity. (b) Aminotransferase reaction catalyzed by the abzyme.

Date: 2016-01-03; view: 766