Structure and Function of Vps15 in the Endosomal G Protein Signaling Pathway ,

G protein-coupled receptors mediate cellular responses to a wide variety of stimuli, including taste, light and neurotransmitters. In the yeast Saccharomyces cerevisiae, activation of the pheromone pathway triggers events leading to mating. The view had long been held that the G protein-mediated signal occurs principally at the plasma membrane. Recently, it has been shown that the G protein α subunit Gpa1 can promote signaling at endosomes and requires two components of the sole phosphatidylinositol-3-kinase in yeast, Vps15 and Vps34. Vps15 contains multiple WD repeats and also binds to Gpa1 preferentially in the GDP-bound state; these observations led us to hypothesize that Vps15 may function as a G protein β subunit at the endosome. Here we show an X-ray crystal structure of the Vps15 WD domain that reveals a seven-bladed propeller resembling that of typical Gβ subunits. We show further that the WD domain is sufficient to bind Gpa1 as well as to Atg14, a potential Gγ protein that exists in a complex with Vps15. The Vps15 kinase domain together with the intermediate domain (linking the kinase and WD domains) also contributes to Gpa1 binding, and is necessary for Vps15 to sustain G protein signaling. These findings reveal that the Vps15 Gβ-like domain serves as a scaffold to assemble Gpa1 and Atg14, whereas the kinase and intermediate domains are required for proper signaling at the endosome

Kinetic mechanism of kanamycin nucleotidyltransferase from Staphylococcus aureus, Bioorg

Kanamycin nucleotidyltransferase (KNTase) catalyzes the transfer of the adenyl group from MgATP to either the 4Ј or 4Љ-hydroxyl group of aminoglycoside antibiotics. The steady state kinetic parameters of the enzymatic reaction have been measured by initial velocity, product, and dead-end inhibition techniques. The kinetic mechanism is ordered where the antibiotic binds prior to MgATP and the modified antibiotic is the last product to be released. The effects of altering the relative solvent viscosity are consistent with the release of the products as the rate-limiting step. The pH profiles for V max and V/K ATP show that a single ionizable group with a pK of ϳ8.9 must be protonated for catalysis. The V/K profile for kanamycin as a function of pH is bell-shaped and indicates that one group must be protonated with a pK value of 8.5, while another group must be unprotonated with a pK value of 6.6. An analysis of the kinetic constants for 10 different aminoglycoside antibiotics and 5 nucleotide triphosphates indicates very little difference in the rate of catalysis or substrate binding among these substrates