Biochemistry

xvii+1361hlm.;29c

Principles of biochemistry

New Yorkxxx, 1.209 p.: Illus.; 28 c

Fundamentals Of Biochemistry: Life At The Molecular Level

Voet, Voet and Pratt’s Fundamentals of Biochemistry, 5e addresses the enormous advances in biochemistry, particularly in the areas of structural biology and Bioinformatics, by providing a solid biochemical foundation that is rooted in chemistry to prepare students for the scientific challenges of the future. While continuing in its tradition of presenting complete and balanced coverage that is clearly written and relevant to human health and disease, Fundamentals of Biochemistry, 5e includes new pedagogy and enhanced visuals that provide a pathway for student learning. The authors are careful to present new information such that it links it to existing content, ever mindful that students assimilate new information only in the proper context

Electrostatic Control Of Enzyme Reactions: The Mechanism Of Inhibition Of Glucose Oxidase By Putrescine

The interaction of putrescine dihydrochloride with glucose oxidase is reported. At pH 7.65 glucose oxidase is strongly anionic (Z = −80). The pKₐ of an essential acidic group on the reduced form of the enzyme is extremely sensitive to ionic strength, as predicted by simple electrostatic theory [J. G. Voet, J. Coe, J. Epstein, V. Matossian, and T. Shipley (1981), Biochemistry, 20, 7182–7185]. Putrescine dihydrochloride was found to inhibit glucose oxidase at pH 7.65 at a constant ionic strength of 0.05. The kinetics do not obey simple competitive inhibition, however. The data can best be explained by a model in which change in the electrostatic potential of the enzyme on putrescine binding changes the observed pKₐ of the essential acidic group. The pH dependence of putrescine inhibition supports this interpretation. At I = 0.05, 5 mM putrescine was found to change the pKₐ of the essential acidic group from 7.6 to 7.1. The shift in the pKₐ as a function of putrescine concentration at pH 7.7 and I = 0.05 also supports the model presented. The Kₐ for putrescine to the active form of the enzyme was calculated to be 4.2 mm

Active Site Generation Of A Protonically Unstable Suicide Substrate From A Stable Precursor: Glucose Oxidase And Dibromonitromethane

Bromonitromethane is an inefficient suicide substrate for glucose oxidase (in contrast to the case of CH₃CCl=NO₂- and D-amino acid oxidase) because, in the enzyme-substrate encounter step, the required ionization states of enzyme (EH₀⁺, pKₐ ~ 3.5) and substrate (CHBr=NO₂-, pKₐ ~ 8.3) cannot be highly populated simultaneously. Because reprotonation of CHBr=NO₂- is rapid at the pH value used for the assay of glucose oxidase, presentation of the enzyme with the preformed anion could not be exploited in this case. We circumvent this difficulty by allowing the enzyme to reductively dehalogenate CHBr₂-NO₂, thereby generating the desired protonically unstable suicide substrate in situ (Eᵣ + CHBr₂NO₂ --\u3e E₀ + CHBr=NO₂- + HBr + H⁺). Irreversible inactivation of the enzyme, because of the formation of a dead-end N-5 formylflavin adduct, is more than 100-fold faster when CHBr=NO₂- is generated in situ than when it is externally applied. The remaining competitive fates of CHBr=NO₂- at the active site are protonation and release or oxidation to HCOBr (or HCONO₂). Strong support for these conclusions comes from (1) the brisk evolution of CH3CBr=NO₂- (which is too bulky to act further as an efficient suicide substrate) from the enzyme-catalyzed reductive debromination of CH₃CBr₂NO₂, (2) the 1:1 stoichiometry of enzyme inactivation, and (3) the identification of the modified flavin as 5-formyl-1,5-dihydro-FAD

5\u27-Azido-N-1-Naphthylphthalamic Acid, A Photolabile Analog Of N-1-Naphthylphthalamic Acid: Synthesis And Binding Properties In Curcurbita Pepo L

A photolabile analog of N-1-naphthylphthalamic acid (NPA), 5′-azido-N-1-naphthylphthalamic acid (Az-NPA), has been synthesized and characterized. This potential photoaffinity label for the plasma membrane NPA binding protein competes with [³H]NPA for binding sites on Curcurbita pepo L. (zucchini) hypocotyl cell membranes with K(0.5) = 2.8 × 10⁻⁷ molar. The K(0.5) for NPA under these conditions is 2 × 10⁻⁸ molar, indicating that the affinity of Az-NPA for the membranes is only 14-fold lower than NPA. While the binding of Az-NPA to NPA binding sites is reversible in the dark, exposure of the Az-NPA treated membranes to light results in a 30% loss in [³H]NPA binding ability. Pretreatment of the membranes with NPA protects the membranes against photodestruction of [³H]NPA binding sites by Az-NPA supporting the conclusion that Az-NPA destroys these sites by specific covalent attachment