To determine the effectiveness of the ribosome as a catalyst, we compared the rate of uncatalyzed peptide bond formation, by the reaction of the ethylene glycol ester of N-formylglycine with Tris(hydroxymethyl)aminomethane, with the rate of peptidyl transfer by the ribosome. Activation parameters were also determined for both reactions, from the temperature dependence of their second-order rate constants. In contrast with most protein enzymes, the enthalpy of activation is slightly less favorable on the ribosome than in solution. The 2 x 10(7)-fold rate enhancement produced by the ribosome is achieved entirely by lowering the entropy of activation. These results are consistent with the view that the ribosome enhances the rate of peptide bond formation mainly by positioning the substrates and/or water exclusion within the active site, rather than by conventional chemical catalysis.

Orotidine 5'-monophosphate decarboxylase has been heavily examined in recent years due to its enzymatic proficiency, which provides a catalytic enhancement to a reaction rate approximately 1017 times greater than that of the nonenzymatic reaction. Several mechanisms proposed to explain this catalytic enhancement have included covalent addition, ylide or carbene formation, and most recently concerted protonation. All of these mechanisms have circumvented the formation of a high-energy vinyl anionic intermediate. To investigate the presence of an anionic intermediate, 13C isotope effect studies have been performed using the alternate substrate 5-fluoro-OMP (OMP = orotidine 5'-monophosphate). Isotope effects obtained for the wild-type enzyme with OMP and 5-fluoro-OMP are 1.0255 and 1.0106, respectively, corresponding to a decrease of approximately 1.5% for 5-fluoro-OMP. With the K59A enzyme, the intrinisic isotope effects show a similar decrease of approximately 1.9% from 1.0543 with OMP to 1.0356 with 5-fluoro-OMP. This decrease results from the inductive effect of the fluorine, which stabilizes the carbanion intermediate by electron withdrawal and produces a reaction with an earlier transition state. The isotope effect for the decarboxylation of the slow substrate 2'-deoxy-OMP produced a intrinsic isotope effect of nearly 1.0461.

There has been some speculation that the C-6 position in UMP may be unusually acidic, stabilizing a carbanion that is generated at this position during OMP decarboxylation. On the basis of the rate of OH- catalyzed deuterium exchange at elevated temperatures we estimate that the pKa value for ionization at C-6 of dimethyl uracil is 34 +/- 2 in water. The same method yields a value of 37 +/- 2 for ionization at C-2 of thiophene in good agreement with the value determined by polarographic methods. The barrier to proton release (46 kcal/mol) is even higher than that for CO2 release from orotic acid derivatives.

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Large‐scale transient expression in mammalian cells is a rapid protein production technology often used to shorten overall timelines for biotherapeutics drug discovery. In this study we demonstrate transient expression in a Chinese hamster ovary (CHO) host (ExpiCHO‐S™) cell line capable of achieving high recombinant antibody expression titers, comparable to levels obtained using human embryonic kidney (HEK) 293 cells. For some antibodies, ExpiCHO‐S™ cells generated protein materials with better titers and improved protein quality characteristics (i.e., less aggregation) than those from HEK293. Green fluorescent protein imaging data indicated that ExpiCHO‐S™ displayed a delayed but prolonged transient protein expression process compared to HEK293. When therapeutic glycoproteins containing non‐Fc N‐linked glycans were expressed in transient ExpiCHO‐S™, the glycan pattern was unexpectedly found to have few sialylated N‐glycans, in contrast to glycans produced within a stable CHO expression system. To improve N‐glycan sialylation in transient ExpiCHO‐S™, we co‐transfected galactosyltransferase and sialyltransferase genes along with the target genes, as well as supplemented the culture medium with glycan precursors. The authors have demonstrated that co‐transfection of glycosyltransferases combined with medium addition of galactose and uridine led to increased sialylation content of N‐glycans during transient ExpiCHO‐S™ expression. These results have provided a scientific basis for developing a future transient CHO system with N‐glycan compositions that are similar to those profiles obtained from stable CHO protein production systems. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2724, 2019