Ribonuclease sites

However, there are a number of other miscellaneous biological roles played by this complex. The [Co(NH3)6]3+ ion has been shown to inhibit the hammerhead ribozyme by displacing a Mn2+ ion from the active site.576 However, [Co(NH3)6]3+ does not inhibit ribonuclease H (RNase),577 topoisomerase I,578 or hairpin ribozyme,579 which require activation by Mg2+ ions. The conclusions from these studies were that an outer sphere complex formation between the enzyme and Mgaq2+ is occuring rather than specific coordination of the divalent ion to the protein. These results are in contrast to DNase I inhibition by the same hexaammine complex. Inhibition of glucose-induced insulin secretion from pancreatic cells by [Co(NH3)6]3+ has been found.580 Intracellular injection of [Co(NH3)6]3+ into a neurone has been found to cause characteristic changes to the structure of its mitochondria, and this offers a simple technique to label neuronal profiles for examination of their ultrastructures.581... 

The methionine 29 is on the outside of ribonuclease-S and simple absorbed [PtClJ2- or [PtC en] should have been rapidly removed from this site. We therefore believe that the platinum complex has reacted... 

When the pH of ribonuclease was raised to 8.0 from 5.5 a second site became partially occupied in which the platinum bound to histidine 119. At the lower pH this group would be protonated and blocked. As stated above for this reason groups such as terminal —NH2, arginines and lysines are not likely to be as effective as thio-ether in displacing chloride from chloride complexes of platinum. (We are grateful to Dr. L. Johnson (Oxford) for supplying us with much of this information). 

Crestfield, A.M., Stein, W.H., and Moore, S. (1963) Alkylation and identification of the histidine residues at the active site of ribonuclease. J. Biol. Chem. 238, 2413-2419. 

Divalent metal ions are essential for ribonuclease H activity. Two Mn(II) ions have been located in the catalytic site of ribonuclease H domain of HIV-1 reverse transcriptase in close proximity to the four acidic residues Asp443, Glu478, Asp498, and Asp549 after soaking crystals in 45 mM MnCl2 (406). 

E. Haas, G. T. Montelione, C. A. McWherter, and H. A. Scheraga, Local structure in a tryptic fragment of performic acid oxidized ribonuclease A corresponding to a proposed polypeptide chain-folding initiation site detected by tyrosine fluorescence lifetime and proton magnetic resonance measurements, Biochemistry 26, 1672-1683 (1987). 

Since the rate constants of bimolecular diffusion-limited reactions in isotropic solution are proportional to T/ these data testify to the fact that the kt values are linearly dependent on the diffusion coefficient D in water, irrespective of whether the fluorophores are present on the surface of the macromolecule (human serum albumin, cobra neurotoxins, proteins A and B of the neurotoxic complex of venom) or are localized within the protein matrix (ribonuclease C2, azurin, L-asparaginase).1 36 1 The linear dependence of the functions l/Q and l/xF on x/t] indicates that the mobility of protein structures is correlated with the motions of solvent molecules, and this correlation results in similar mechanisms of quenching for both surface and interior sites of the macromolecule. 

An example of this effect is provided by ribonuclease A (RNase A). At pH 8 and 37°, the rate of deamidation of Asn67 was more than 30-fold lower in the native than in the unfolded protein [111]. Deamidation of the native RNase A was also ca. 30-fold slower than of an octapeptide whose sequence is similar to that of the deamidation site, although the reaction mechanisms were similar [108][123],... 

In the mechanism of the pancreatic hydrolase ribonuclease, a specialized histidine within the active site acts as a general acid or proton donor to begin cleavage of the phosphodiester linkage of the substrate RNA. 

The pH-rate profile for the action of the enzyme shows a typical pH maximum, with sharply lower rates at either higher or lower pH than the optimum these facts suggest that both an acidic and a basic group are required for activity (Herries, 1960). The two essential histidine residues could serve as these groups if, in the active site, one were protonated and the other present in its basic form. The simultaneous acid-base catalysis would parallel that of the model system (discussed below) of Swain and J. F. Brown. The essential lysine, which binds phosphate, presumably serves to bind a phosphate residue of the ribonucleic acid. These facts led Mathias and coworkers to propose the mechanism for the action of ribonuclease that is shown in (13) (Findlay et al., 1961). 

For ribonuclease A the occurrence of conformational changes and the occurrence of acid-base catalysis has been well documented. The overall mechanism can be envisaged as follows. The enzyme exists in dynamic equilibrium between two forms differing in the structure of the active site groove. The substrate is bound almost as rapidly as it can diffuse to the active site. Binding of the substrate induces a conformational change that... 

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