Rapid Ribonuclease P Kinetics Measured by Stopped-Flow Fluorescence and Fluorescence Anisotropy.

Stopped-flow fluorescence spectroscopy is a highly sensitive method for measuring rapid enzyme kinetics. A wide range of fluorophores can be employed, and fluorescence and fluorescence polarization can be measured. Thus, binding, conformational changes, and catalysis can, in principle, be measured, making it helpful in probing the entire kinetic landscape of a reaction. In this chapter, we use the bacterial RNA processing enzyme ribonuclease P (RNase P) as a model system to illustrate the determination of the kinetic constants for substrate binding and cleavage, thus allowing mechanistic questions regarding the effects of reaction conditions, mutations, or drug binding to be answered.

The specificity landscape of bacterial ribonuclease P.

Developing quantitative models of substrate specificity for RNA processing enzymes is a key step toward understanding their biology and guiding applications in biotechnology and biomedicine. Optimally, models to predict relative rate constants for alternative substrates should integrate an understanding of structures of the enzyme bound to "fast" and "slow" substrates, large datasets of rate constants for alternative substrates, and transcriptomic data identifying in vivo processing sites. Such data are either available or emerging for bacterial ribonucleoprotein RNase P a widespread and essential tRNA 5' processing endonuclease, thus making it a valuable model system for investigating principles of biological specificity. Indeed, the well-established structure and kinetics of bacterial RNase P enabled the development of high throughput measurements of rate constants for tRNA variants and provided the necessary framework for quantitative specificity modeling. Several studies document the importance of conformational changes in the precursor tRNA substrate as well as the RNA and protein subunits of bacterial RNase P during binding, although the functional roles and dynamics are still being resolved. Recently, results from cryo-EM studies of E. coli RNase P with alternative precursor tRNAs are revealing prospective mechanistic relationships between conformational changes and substrate specificity. Yet, extensive uncharted territory remains, including leveraging these advances for drug discovery, achieving a complete accounting of RNase P substrates, and understanding how the cellular context contributes to RNA processing specificity in vivo.

Substrate Specificity and Chemical Mechanism for the Reaction Catalyzed by Glutamine Kinase.

Campylobacter jejuni, a leading cause of gastroenteritis worldwide, has a unique O-methyl phosphoramidate (MeOPN) moiety attached to its capsular polysaccharide. Investigations into the biological role of MeOPN have revealed that it contributes to the pathogenicity of C. jejuni, and this modification is important for the colonization of C. jejuni. Previously, the reactions catalyzed by four enzymes (Cj1418-Cj1415) from C. jejuni that are required for the biosynthesis of the phosphoramidate modification have been elucidated. Cj1418 (l-glutamine kinase) catalyzes the formation of the initial phosphoramidate bond with the ATP-dependent phosphorylation of the amide nitrogen of l-glutamine. Here we show that Cj1418 catalyzes the phosphorylation of l-glutamine through a three-step reaction mechanism via the formation of covalent pyrophosphorylated ( Enz-X-Pß-Pγ) and phosphorylated ( Enz-X-Pß) intermediates. In the absence of l-glutamine, the enzyme was shown to catalyze a positional isotope exchange (PIX) reaction within ß-[18O4]-ATP in support of the formation of the Enz-X-Pß-Pγintermediate. In the absence of ATP, the enzyme was shown to catalyze a molecular isotope exchange (MIX) reaction between l-glutamine phosphate and [15N-amide]-l-glutamine in direct support of the Enz-X-Pßintermediate. The active site nucleophile has been identified as His-737 based on the lack of activity of the H737N mutant and amino acid sequence comparisons. The enzyme was shown to also catalyze the phosphorylation of d-glutamine, γ-l-glutamyl hydroxamate, γ-l-glutamyl hydrazide, and ß-l-aspartyl hydroxamate, in addition to l-glutamine.