Histidine Residues Of Ribonuclease Research Articles

Effect of mutagenesis at each of five histidine residues on enzymatic activity and stability of ribonuclease H from Escherichia coli.

To examine the role of histidine residues in ribonuclease H from Escherichia coli, kinetic parameters for the enzymatic activity and conformational stabilities against guanidine hydrochloride denaturation of mutant enzymes, in which each of the five histidine residues was replaced with alanine, were determined and compared with the wild-type enzyme. The mutation of His83 resulted in a marked increase in Km along with an increase in kcat. The mutation of His114 caused a large reduction in both the free energy of unfolding in water, delta GH2O, and the mid-point of the unfolding curve, [D]1/2. These results indicate that His83, which is one of the four well-exposed histidine residues in the crystal structure, is located close to a substrate-binding site, and His114, which is buried inside the protein molecule, contributes to the conformational stability, probably through the formation of a hydrogen bond with a main-chain carbonyl group. None of the histidine residues is required for activity.

Local secondary structure in ribonuclease A denatured by guanidine · HCl near 1 °C

Recent work has shown that a short α-helix can be stable in water near 1 °C when stabilized by specific interactions between side-chains, while earlier “host-guest” results with random copolymers have shown that a short α-helix is unstable in water at all temperatures in the absence of stabilizing side-chain interactions. As regards the mechanism of protein folding, it is now reasonable on energetic grounds to consider isolated α-helices and β-sheets as the first intermediates on the pathway of protein folding. Proton nuclear magnetic resonance is used here to detect isolated secondary structures in ribonuclease A denatured by guanidine · HCl (GuHCl). Temperatures near 1 °C are used because the low-temperature stability of the C-peptide helix may be a general property of isolated secondary structures in water. Our procedure is to titrate with GuHCl the C2H resonance lines of the four histidine residues of denatured ribonuclease A. Studies of model peptides (C-peptide (lactone) and C-peptide carboxylate, residues 1 to 13 of ribonuclease A; S-peptide, residues 1 to 20) show linear titration curves for the C2H resonance of His12 above 0.5 M-GuHCl, once helix unfolding is complete. Deviations from this line are used to monitor helix formation. The GuHCl titration curves of the other three histidine residues are also linear, once unfolding is complete. The results show that the helix found in C-peptide and S-peptide is also found in denatured ribonuclease A, where it behaves as an isolated helix not stabilized significantly by interactions with other chain segments. Studies of denatured S-protein show that the remaining three His residues, His48, His105 and His119, are involved in structure only below 1 m-GuHCl at 9 °C, pH 1.9. The nature of this structure is not known. The main conclusion from this work is that the His12 helix can be observed as a stable, isolated helix in denatured ribonuclease A near 1 °C, and that none of the other three His residues is involved in a comparably stable local structure. In native ribonuclease A, His12 is within an α-helix and the other three His residues are involved in a 3-stranded β-sheet structure. The helix-coil transition of C-peptide has also been studied for other side-chain resonances by GuHCl titration. Typically, but not always, the titration curves are linear after helix unfolding takes place and resonance lines from different residues of the same amino acid type can be resolved in GuHCl solutions. This is true of the four histidine residues of ribonuclease A although their p K values in 5 m-GuHCl are nearly the same. In C-peptide, the βCH 3 resonance of Ala6 is affected strongly by GuHCl while the lines of Ala4 and Ala5 are shifted only weakly by GuHCl. Evidently the interactions between GuHCl and side-chains in an unfolded peptide depend upon neighboring groups.

The pentaammineruthenium(III)-histidine complex in ribonuclease A as an optical probe of conformational change

The formation of stable transition metal complexes between pentaammineruthenium(III) and the imidazole moiety of histidine residues in ribonuclease A is described. Studies of the kinetics of incorporation suggest that three of the four histidines are reactive and ion-exchange chromatography affords the isolation of three derivatives containing a single ruthenium-histidine complex. Equilibrium studies of the thermal- and urea-induced unfolding of one of these derivatives show that the ligand-to-metal charge-transfer absorptions above 300 nm can be used to follow the conformational changes in the vicinity of the complex. For the urea-induced unfolding of this derivative, the coincidence of the transition curves obtained from the charge-transfer absorptions and from difference ultraviolet spectroscopy at 286 nm, which measures the exposure of buried tyrosine residues to solvent, suggest that only two conformations, native and unfolded, are present at equilibrium in significant concentrations.

Proton magnetic resonance studies of ribonuclease T 1. Assignment of histidine-40 peak and analysis of the active site

Nuclear magnetic resonances of the C-2 protons of the three histidine residues in ribonuclease T 1 have been studied at 360 MHz as a function of pH to discuss the structure of the active site. Comparison of the order of deuterium exchange of the histidine peaks with tritium incorporation rates into individual histidines of the enzyme leads to the unambigous assignment of one of the C-2 proton peaks to histidne-40. It has been concluded that histidine-40 is in the active site, interacting with a charged group of pK 4.1, which is replaced by the phosphate group of guanosine-3′-monophosphate in the enzyme-inhibitor complex. Histidine-92 is most likely a binding site for the complex, where the existence of a hydrogen bond between N-7 of the inhibitor and the ring NH proton of the histidine is suggested on the basis of NMR data.

Correlation proton magnetic resonance studies at 250 MHz of bovine pancreatic ribonuclease. I. Reinvestigation of the histidine peak assignments

The deuterium exchange kinetics of the C(2) protons of the four histidine residues of native bovine pancreatic ribonuclease A have been followed at pH 6.5 and 8.0 by proton magnetic resonance spectroscopy (1H NMR). Comparison of the order of exchange of the histidine peaks with tritium exchange rates into individual histidine residues [Ohe, M., Matsuo, H., Sakiyama, F., and Narita, K. (1974), J. Biochem. (Tokyo) 75, 1197] supports the previous assignment of histidine NMR peaks H(1) and H(4) to histidine-105 and histidine-48 but requires reassignment of peaks H(2) and H(3) to histidine-119 and histidine-12, respectively. Ribonuclease A samples having differentially deuterated histidines have been used to verify the existence of crossover points in the histidine proton magnetic resonance titration curves and to observe the discontinuous titration curve of histidine-48. Proton magnetic resonance peaks have been assigned to the C(4) protons of the four histidine residues of ribonuclease A on the basis of their unit proton areas and by matching their titration shifts with the more readily visible C(2)-H peaks of the histidines. The pK' values derived from the C(4)-H data agree, within experimental limits, with those derived from C(2)-H data. The C(4)-H peaks were assigned to histidine-12, -48, -105, and -119 of ribonuclease A on the basis of their pH dependence, pK' values, shifts of their pK' values in the presence of inhibitor cytidine 3'-phosphate, and by comparison with the assignments of the histidine C(2)-H peaks above.

Inactivation of pancreatic ribonuclease with hydroxylamine-oxygen-cupric ion

In the studies of ribonuclease, it was found that a system containing hydroxylamine, Cn 2+ and O 2 will inactivate its biological activity. All three components are essential for the inactivation. The inactivation of ribonuclease by this system is pH-dependent. The rate of inactivation was studied as a function of hydroxylamine concentration and Cu 2+ concentration. It is interesting to note that both Tris and phosphate serve as competitive inhibitors. It was also found that all four histidine residues of ribonuclease were missing when the inactivated ribonuclease was subjected to acid hydrolysis followed by amino acid analysis.

Structural Studies of Ribonuclease. XXIV. The Application of Nuclear Magnetic Resonance Spectroscopy to Distinguish between the Histidine Residues of Ribonuclease1

Structural Studies of Ribonuclease. XXIV. The Application of Nuclear Magnetic Resonance Spectroscopy to Distinguish between the Histidine Residues of Ribonuclease¹

States of amino acid residues in proteins: IX. Histidine residues in ribonuclease in the presence of cytidine- and uridine-3′-phosphates as observed with diazonium-1-H-tetrazole

Diazonium-1-H-tetrazole (DHT), a new coupling reagent for histidine residues, was reacted with bovine pancreatic ribonuclease (RNase) in the presence and absence of nucleotides; cytidine-3′-phosphate (CMP), uridine-3′-phosphate(UMP), adenosine-3′-phosphate (AMP), and guanosine-3′-phosphate (GMP). Coupling to the four histidine residues in the native RNase molecule in the absence of nucleotide proceeds in four steps with increasing DHT concentration, such that each of these four residues is coupled at a different DHT concentration. The reactivities were affected by the presence of CMP or UMP, a product of the RNase-catalyzed reaction, whereas AMP or GMP, which are not products of the reaction, show no effect on the reactivities. The effect of CMP is different from that of UMP; CMP enhances the reactivity of a residue which is the third to be coupled in the absence of nucleotide, while UMP enhances the reactivity of a residue which is the second or fourth to be coupled. The possible participation in the enzymic activity of these two histidine residues with different reactivities and their different responses to nucleotides were thus demonstrated.