Through kinetic studies we provide a quantitative reactivity scale for ten electrophilic fluorination reagents.

Methods for the incorporation of non-natural amino acids into proteins have advanced significantly over recent years and in this tutorial review we aim to give a general overview of the area. These techniques offer the possibility of modulating the structures and functions of proteins and thus permit the generation of novel designed systems for both biocatalytic and mechanistic studies. Four complementary approaches are discussed in detail along with examples of their application. The advantages and disadvantages of each technique are also discussed.

Carbon dioxide is fundamental to the physiology of all organisms. There is considerable interest in the precise molecular mechanisms that organisms use to directly sense CO 2 . Here we demonstrate that a mammalian recombinant G-protein-activated adenylyl cyclase and the related Rv1625c adenylyl cyclase of Mycobacterium tuberculosis are specifically stimulated by CO 2 . Stimulation occurred at physiological concentrations of CO 2 through increased k cat . CO 2 increased the affinity of enzyme for metal co-factor, but contact with metal was not necessary as CO 2 interacted directly with apoenzyme. CO 2 stimulated the activity of both G-protein-regulated adenylyl cyclases and Rv1625c in vivo. Activation of G-protein regulated adenylyl cyclases by CO 2 gave a corresponding increase in cAMP-response element-binding protein (CREB) phosphorylation. Comparison of the responses of the G-protein regulated adenylyl cyclases and the molecularly, and biochemically distinct mammalian soluble adenylyl cyclase revealed that whereas G-protein-regulated enzymes are responsive to CO 2 , the soluble adenylyl cyclase is responsive to both CO 2 and bicarbonate ion. We have, thus, identified a signaling enzyme by which eukaryotes can directly detect and respond to fluctuating CO 2 .

The Slr1991 adenylyl cyclase of the model prokaroyte Synechocystis PCC 6803 was stimulated 2-fold at 20 mM total C(i) (inorganic carbon) at pH 7.5 through an increase in k(cat). A dose response demonstrated an EC50 of 52.7 mM total C(i) at pH 6.5. Slr1991 adenylyl cyclase was activated by CO2, but not by HCO3-. CO2 regulation of adenylyl cyclase was conserved in the CyaB1 adenylyl cyclase of Anabaena PCC 7120. These adenylyl cyclases represent the only identified signalling enzymes directly activated by CO2. The findings prompt an urgent reassessment of the activating carbon species for proposed HCO3--activated adenylyl cyclases.