Ionization constants and thermodynamic parameters of histidine and derivatives

Kriegl

Journal of Biological Chemistry

2004

132

We have examined the effects of active site residues on ligand binding to the heme iron of mouse neuroglobin using steady-state and time-resolved visible spectroscopy. Absorption spectra of the native protein, mutants H64L and K67L and double mutant H64L/K67L were recorded for the ferric and ferrous states over a wide pH range (pH 4 -11), which allowed us to identify a number of different species with different ligands at the sixth coordination, to characterize their spectroscopic properties, and to determine the pK values of active site residues. In flash photolysis experiments on CO-ligated samples, reaction intermediates and the competition of ligands for the sixth coordination were studied. These data provide insights into structural changes in the active site and the role of the key residues His 64 and Lys 67 . His 64 interferes with exogenous ligand access to the heme iron. Lys 67 sequesters the distal pocket from the solvent. The heme iron is very reactive, as inferred from the fast ligand binding kinetics and the ability to bind water or hydroxyl ligands to the ferrous heme. Fast bond formation favors geminate rebinding; yet the large fraction of bimolecular rebinding observed in the kinetics implies that ligand escape from the distal pocket is highly efficient. Even slight pH variations cause pronounced changes in the association rate of exogenous ligands near physiological pH, which may be important in functional processes.

Section: Resultsmentioning

confidence: 99%

Structural Dynamics in the Active Site of Murine Neuroglobin and Its Effects on Ligand Binding

Kriegl

Journal of Biological Chemistry

2004

132

Journal of Biological Chemistry

“…The histidine derivative, N,N-dimethylhistidine (His(Me) 2 ), in which both of the imidazole nitrogens are methylated, was synthesized as described previously (13). Nova Syn TGR resin (NovaBiochem) was used, and all couplings were performed with O-benzotriazol-1-yl-N,N,NЈ,NЈ-tetramethyluronium tetrafluoroborate/1-hydroxybenzotriazole using N-methyl morpholine as base.…”

Section: Methodsmentioning

confidence: 99%

Studies on the Subsite Specificity of Rat Nardilysin (N-Arginine Dibasic Convertase)

Chow¹,

Csuhai²,

Juliano³

et al. 2000

Self Cite

The subsite specificity of rat nardilysin was investigated using fluorogenic substrates of the type 2-aminobenzoyl-GGX 1 X 2 RKX 3 GQ-ethylenediamine-2,4-dinitrophenyl, where P 2 , P 2 , and P 3 residues were varied. (The nomenclature of Schechter and Berger (Schechter, I., and Berger, A. (1967) Biochem. Biophys. Res. Commun. 27, 157-162) is used where cleavage of a peptide occurs between the P 1 and P 1 residues, and adjacent residues are designated P 2 , P 3 , P 2 , P 3 , etc.) There was little effect on K m among different residues at any of these positions. In contrast, residues at each position affected k cat , with P 2 residues having the greatest effect. The S 3 , S 2 , and S 2 subsites differed in their amino acid preference. Tryptophan and serine, which produced poor substrates at the P 2 position, were among the best P 2 residues. The specificity at P 3 was generally opposite that of P 2 . Residues at P 2 , and to a lesser extent at P 3 , influenced the cleavage site. At the P 2 position, His, Phe, Tyr, Asn, or Trp produced cleavage at the amino side of the first basic residue. In contrast, a P 2 Ile or Val produced cleavage between the dibasic pair. Other residues produced intermediate effects. The pH dependence for substrate binding showed that the enzyme prefers to bind a protonated histidine. A comparison of the effect of arginine or lysine at the P 1 or P 1 position showed that there is a tendency to cleave on the amino side of arginine and that this cleavage produces the highest k cat values. Nardilysin (N-arginine dibasic convertase, EC 3.4.24.61) is a 130-kDa metallopeptidase and a member of the relatively newly discovered pitrilysin family of metallopeptidases, also referred to as the inverzincins. An active site zinc binding motif, HXXEH, which is inverted relative to the more common HEXXH motif (1), characterizes this family of metallopeptidases. The geometry and mechanism of peptide cleavage at an inverted zinc-binding site has not been elucidated. Aside from nardilysin, three other members of the pitrilysin family have been identified: insulin-degrading enzyme (EC 3.4.24.56) (2, 3), protease III from E. coli (EC 3.4.24.55) (4), and a recently described human metallopeptidase (5).Nardilysin is unique in that it contains an acidic region, which, depending on the particular species, is composed of 43 (human) to 59 (mouse) glutamate and aspartate residues within a 76-amino acid stretch. It has been suggested that this acidic domain might play a role in the regulation of nardilysin activity by forming charge-charge complexes with other cellular components (6, 7).The initial characterization of the specificity of nardilysin led to the conclusion that the enzyme cleaves peptide substrates on the amino side of an arginine residue of paired basic residues (8 -10). It has been found that although the enzyme cleaves a number of peptides between paired dibasic residues of the type Arg-Arg (dynorphin A, BAM12 residues 1-8) and Arg-Lys (␣-neoendorphin), with somatostatin 28 as substrate cleavage occurs ...

“…Upon losing a proton, the imidazolium cation (Scheme 1 A) produces two different tautomers of imidazole: that with a free N61 site (Scheme 1 B; see Scheme 1 D for atom nomenclature) and that with a free NE2 site (Scheme 1 C). Studies of histidine methylated at either the NE2 or the N61 atom have shown that the two sites have distinct microscopic binding constants -for the proton, NE2 being -0.6 pH unit more basic than N61 (Reynolds et al, 1973;Boschcov et al, 1983;Tanokura, 1983). As listed in Table 1, the difference in pKa values has both enthalpic (N-H bond strength) and entropic (accessibility to solvent) components (Boschcov et al, 1983).…”

Section: Introductionmentioning

confidence: 99%

“…Studies of histidine methylated at either the NE2 or the N61 atom have shown that the two sites have distinct microscopic binding constants -for the proton, NE2 being -0.6 pH unit more basic than N61 (Reynolds et al, 1973;Boschcov et al, 1983;Tanokura, 1983). As listed in Table 1, the difference in pKa values has both enthalpic (N-H bond strength) and entropic (accessibility to solvent) components (Boschcov et al, 1983). The latter emphasizes the role of solvation in modulating the properties of the imidazole group.…”

Section: Introductionmentioning

confidence: 99%

The tautomeric state of histidines in myoglobin

Bhattacharya

Sukits

Maclaughlin

et al. 1997

Biophysical Journal

1H-15N HMQC spectra were collected on 15N-labeled sperm whale myoglobin (Mb) to determine the tautomeric state of its histidines in the neutral form. By analyzing metaquoMb and metcyanoMb data sets collected at various pH values, cross-peaks were assigned to the imidazole rings and their patterns interpreted. Of the nine histidines not interacting with the heme in sperm whale myoglobin, it was found that seven (His-12, His-48, His-81, His-82, His-113, His-116, and His-119) are predominantly in the N epsilon2H form with varying degrees of contribution from the Ndelta1 H form. The eighth, His-24, is in the Ndelta1H state as expected from the solid state structure. 13C correlation spectra were collected to probe the state of the ninth residue (His-36). Tentative interpretation of the data through comparison with horse Mb suggested that this ring is predominantly in the Ndelta1H state. In addition, signals were observed from the histidines associated with the heme (His-64, His-93, and His-97) in the 1H-15N HMQC spectra of the metcyano form. In several cases, the tautomeric state of the imidazole ring could not be derived from inspection of the solid state structure. It was noted that hydrogen bonding of the ring was not unambiguously reflected in the nitrogen chemical shift. With the experimentally determined tautomeric state composition in solution, it will be possible to broaden the scope of other studies focused on the electrostatic contribution of histidines to the thermodynamic properties of myoglobin.