Enzymes lifetime determinants

The chemical shift of the phosphorus resonance of various nucleotides has been studied as a function of pH in the presence and absence of RNase A. The signal shifts upheld on protonation of the phosphate and the apparent pATa of the phosphate group in 2 -CMP complex with RNase is the same as the pATa of histidine-119 in this enzyme as determined by n.m.r. From n.m.r. relaxation rates for the ternary complex manganese(n)-phosphate-E. coli alkaline phosphatase, it has been concluded that an outer-sphere complex is formed which has a shorter lifetime than the enzyme turnover rate. The latter conclusion is consistent with the participation of the complex in the enzymic reaction. 

Proteins start out life as a bunch of amino acids linked together in a head-to-tail fashion—the primary sequence. The one-dimensional information contained in the primary amino acid sequence of cellular proteins is enough to guide a protein into its three-dimensional structure, to determine its specificity for interaction with other molecules, to determine its ability to function as an enzyme, and to set its stability and lifetime. 

While new data acquisition techniques under development may significantly reduce the time and increase the precision of recording diffraction patterns, it is obvious that X-ray diffraction techniques will be restricted to the study of conformations and intermediates which are stable for periods that exceed the normal half-life of these transient species. It is therefore necessary to increase the lifetime of such species so that their three-dimensional structures may be determined in the same manner as a native enzyme. 

In case of local gastrointestinal treatments nucleic acid drugs shall be taken up by enterocytes, usually through endocytosis. The route of uptake determines, subsequently, nucleic acid trafficking and lifetime in the cell. Endocytosis is a multistep process involving binding, internalization, formation of endosomes, fusion with lysosomes, and lysis. The low pH and enzymes within... 

The binding of CO to Cug+ was discovered by low-temperature Fourier transform infrared (FTIR) spectroscopy at 2062 cm (Alben et al., 1981), which was similar to the C-O stretch band of hemocyanin-CO complexes (Fager and Alben, 1972). The band was obtained by flash photolysis of the CO-bound enzyme at liquid nitrogen temperature. The 2062-cm band was stable below 140 K. Subsequently, transient binding of CO as well as O2 to Cu + was also observed at ambient temperature by visible-Soret rapid reaction measurements (Woodruff et al., 1991 Blackmore et al., 1991). The lifetime of Cub-CO at ambient temperature was determined to be about 1.5 ps by transient infrared... 

The mode of immobilization, as well as the source and extent of purification of the enzyme, are important factors in determining the lifetime of the bio-catalyst. Generally, the lifetime of a soluble enzyme electrode is about one week or 25-50 assays, and the physically entrapped polyacrylamide electrodes are satisfactory for about 50-100 assays, depending primarily on the degree of care exercised in the preparation of the polymer. The chemically attached enzyme can be kept for years, if used infrequendy. In frequent use, the GOD electrode has a lifetime of over one year and can be used for over 1000 assays. For 1-amino acid oxidase or uricase (100) biosensors, about 200-1000 assays per electrode can be obtained, depending on the immobilization technique. 

Figure 4 A schematic representation of the experimentai approach for time-resoived XAS measurements. XAS provides local structural and electronic information about the nearest coordination environment surrounding the catalytic metal ion within the active site of a metalloprotein in solution. Spectral analysis of the various spectral regions yields complementary electronic and structural information, which allows the determination of the oxidation state of the X-ray absorbing metal atom and precise determination of distances between the absorbing metal atom and the protein atoms that surround it. Time-dependent XAS provides insight into the lifetimes and local atomic structures of metal-protein complexes during enzymatic reactions on millisecond to minute time scales, (a) The drawing describes a conventional stopped-flow machine that is used to rapidly mix the reaction components (e.g., enzyme and substrate) and derive kinetic traces as shown in (b). (b) The enzymatic reaction is studied by pre-steady-state kinetic analysis to dissect out the time frame of individual kinetic phases, (c) The stopped-flow apparatus is equipped with a freeze-quench device. Sample aliquots are collected after mixing and rapidly froze into X-ray sample holders by the freeze-quench device, (d) Frozen samples are subjected to X-ray data collection and analysis.

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