When hyaluronan synthase from Pasteurella multocida was expressed in E. coli, the bacteria spun out a capsule of hyaluronan. This reaction was analyzed using purified membrane fragments containing the hyaluronan synthase, a radio-labeled tetrasaccharide acceptor (Part A), along with the activated forms of the sugar subunits: UDP-N-acetylglucosamine and UDP-glucuronic acid. In short incubations with the complete mixture, the tetrasaccharide primer elongated by several units, although only odd-numbered chains were visible (Part B lane 2). If one or both activated sugars were left out, the results shown in lanes 3-5 (Part B) were obtained. Why do you suppose that only odd-number chains are visible in your assay? (A) ACCEPTOR nonreducing end HO HO- OH COO™ O glucuronic acid (GICUA) HO- CH3 C=O NH CH₂ T OH O N-acetylglucosamine (GlcNAc) COO но- HO OH OH CH₂ reducing end NH T C=O CH₂ Choose one: O A. Synthase adds GlcNAc much more slowly than it adds GICUA. O B. One of the monosaccharide subunits (GlcNAc) is not labeled. OH O C. Addition of GICUA removes the radio label from the acceptor. O D. The enzyme adds only disaccharide units to the growing chain. (B) BIOSYNTHESIS number of residues 57913 origin |||| markers + GlcNAc + GICUA + GlcNAc 1 2 3 4 lane number (Part B from P.L. DeAngelis, J. Biol. Chem. 274:26557-26562, 1999. With permission from American Society for Biochemistry and Molecular Biology.) + GICUA 5 none

Draw the catalytic triad of a serine protease at the first tetrahedryl intermediate stage.  Your drawing should include the amino acids that make up the triad, any part of the substrate that should be present, and any other molecules that are present in the active site.

During glycogen synthesis, glucose-1P is converted into a molecule called UDPG. This reaction also cleaves uridine triphosphate (UTP) forming uridine monophosphate and pyrophosphate (PPi). Provide four reasons why UTP can be used to power this reaction (no diagrams necessary).

serine proteases act via a two-step catalytic mechanism. However, as a critical scientist, you may want to see data that supports this claim. One experiment that can provide such evidence is called a 'pre-steady state' kinetics experiment using the chromogenic substrate, N-acetyl- phenylalanine p-nitrophenyl ester. As shown below when this substrate is cleaved, the p-nitrophenylate is a yellow product whose absorbance can be measured in real time using a spectrophotometer. When the chromogenic substrate is rapidly mixed together with a limiting amount of protease enzyme, the accumulation of product is measured right away on fast (millisecond) timescales. In this experiment, one observes two distinct phases of the reaction as shown in the plot below. Based upon what you know about the mechanism of serine proteases, provide a mechanistic interpretation for the observation of these two phases of the reaction. + H₂O N-Acetyl-L-phenylalanine p-nitrophenyl ester p-Nitrophenolate Absorbance…