R.J. Hilmoe

Bio: R.J. Hilmoe is an academic researcher from National Institutes of Health. The author has contributed to research in topics: Purine metabolism & Purine riboswitch. The author has an hindex of 3, co-authored 3 publications receiving 412 citations.

Natural Configuration of the Purine Nucleotides in Ribonucleic Acids: Enzymatic Splitting of Purine Internucleotide Linkages

Abstract: Natural Configuration of the Purine Nucleotides in Ribonucleic Acids: Enzymatic Splitting of Purine Internucleotide Linkages

Application of a Theory of Enzyme Specificity to Protein Synthesis

Abstract: The suggestion of Lipmann' that the energy required to drive this reaction to the right came from adenosine triphosphate has been supported by the extensive work2 which has been discussed by the previous speakers on this symposium. While the detailed mechanism of this energy transfer has not been fully elucidated, the ATP apparently forms an amino acid anhydride. The fact that an anhydride of relatively high-energy content is the method of driving the reaction thermodynamically fits in with organic practice in which acyl anhydrides are the usual reagents for acylations. In addition to thermodynamic activation, there is a needed \"kinetic activation.\" For example, the acyl phosphates and acyl adenylates acylate amines non-enzymatically at appreciable rates in aqueous solution,3 but this reaction is certainly not rapid enough or selective enough to account for protein synthesis. The reaction of these acyl anhydrides to form peptides must be catalyzed, and it is this catalysis which is the subject of this paper. An analysis of why this linking of amino acids presents such formidable difficulties is revealing. The difficulty appears to be caused by a combination of requirements, each of which, taken singly, is rather easily satisfied. The first requirement is that an individual position in the protein be occupied by one and only one amino acid. This by itself is not a difficult condition to satisfy, since enzymatic reactions of equally high specificity are well known. The second requirement is that a highmolecular-weight polymer be produced. Again, this, by itself, is not a unique condition, since the enzymatic formation of high-molecular-weight molecules from a given monomer is also familiar, e.g., carbohydrate polymerization catalyzed by phosphorylases. Finally, the macromolecule is formed from many different monomer units, but this requirement is also easily achieved if the units are randomly arranged, e.g., polynucleotide formation by polynucleotide phosphorylase.4 However, the combined requirements, i.e., the synthesis of a macromolecule from many individual monomers to give a single specified sequence, is a problem of different magnitude. A mechanism involving an individual enzyme for each bond would be acceptable from the specificity point of view, but it would require an inconceivably large number of enzymes to form all the proteins of the cell. Moreover, there are other observed conditions, e.g., the necessity of feeding all the amino acids

Assays for differentiation of glutathione S-transferases.

Abstract: Publisher Summary This chapter provides the spectrophotometric, titrimetric, nitrite, and cyanide assay for the differentiation of glutathione S-transferases. Spectrophotometric assays depend upon a direct change in the absorbance of the substrate when it is conjugated with glutathione (GSH). Because each of the reactions is catalyzed at a finite rate in the absence of enzyme, care is needed to reduce nonenzymatic catalysis by minimizing substrate concentrations and by decreasing pH wherever necessary. Titrimetric assay is based on the principle that the conjugation of alkyl halides with GSH can be measured titrimetrically. Although acid production accompanies many of the transferase catalyzed reactions in which thioethers are formed, titrimetry is only used when more convenient assays are not available. Nitrite assay is based on the principle that nitrite is released when GSH reacts with nitroalkanes or with organic nitrate esters. The nitrite can be assayed as the limiting factor in a diazotization reaction with sulfanilamide that produces a readily quantitatable pink dye. Cyanide assay is based on the fact that when glutathione transferases catalyze the attack of the glutathione thiolate ion on the electrophilic sulfur atom of several organic thiocyanates, it results in the formation of an asymmetric glutathionyl disulfide and cyanide. Cyanide can be readily quantitated by a calorimetric method.

The Glutathione S‐Transferases: A Group of Multifunctional Detoxification Proteins

Abstract: The physiological roles of the glutathione S-transferases, by whatever name, seem to result in detoxification As is true of albumin, members of this group of proteins bind an enormous number of compounds that appear to have in common only hydrophobic topography; the binding of bilirubin is an example of a major function common to all higher species If the ligand bears a sufficiently electrophilic center, it will be attacked by the nucleophile GSH; such compounds would be the substrates of the enzyme And should such a ligand be extraordinarily reactive--as, for example, some of the epoxide carcinogens generated by the cytochrome P450-linked, mixed-function oxidases, or even 1-chloro-2,4-dinitrobenzene--then reaction may occur either with GSH or irreversibly with the transferase itself By reason of the wide distribution and high intracellular concentration of these proteins, there appears to be sufficient enzyme for all three roles in detoxification