Water zinc-bound

Water soluble protein with a relative molecular mass of ca. 32600, which particularly contains copper and zinc bound like chelate (ca. 4 gram atoms) and has superoxide-dismutase-activity. It is isolated from bovine liver or from hemolyzed, plasma free erythrocytes obtained from bovine blood. Purification by manyfold fractionated precipitation and solvolyse methods and definitive separation of the residual foreign proteins by denaturizing heating of the orgotein concentrate in buffer solution to ca. 65-70 C and gel filtration and/or dialysis. 

As an illustration, we briefly discuss the SCC-DFTB/MM simulations of carbonic anhydrase II (CAII), which is a zinc-enzyme that catalyzes the interconversion of CO2 and HCO [86], The rate-limiting step of the catalytic cycle is a proton transfer between a zinc-bound water/hydroxide and the neutral/protonated His64 residue close to the protein/solvent interface. Since this proton transfer spans at least 8-10 A depending on the orientation of the His 64 sidechain ( in vs. out , both observed in the X-ray study [87]), the transfer is believed to be mediated by the water molecules in the active site (see Figure 7-1). To carry out meaningful simulations for the proton transfer in CAII, therefore, it is crucial to be able to describe the water structure in the active site and the sidechain flexibility of His 64 in a satisfactory manner. 

Figure 7-2. Properties of CAII active site in the COHH state (zinc-bound hydroxide and protonated His 64). (a) Superposition of a few key residues from two stochastic boundary SCC-DFTB/MM simulations with the X-ray structure [87] (colored based on atom-types) the two sets of simulations did not have any cut-off for the electrostatic interactions between SCC-DFTB and MM atoms but used different treatments for the electrostatic interactions among MM atoms group-based extended electrostatics (in yellow) and atom-based force-shift cut-off (in green). Extended electrostatics simulations sampled configurations with the protonated His 64 too close to the zinc moiety while force-shift simulations consistently sampled the out configuration of His 64 in multiple trajectories, (b) Statistics for productive water-bridges (only from two and four shown here) between the zinc bound water and His 64 with different electrostatics protocols...

Another property relevant to the current discussion is the distribution of water in the active site. Specifically, we characterize the population of various water wires connecting the zinc-bound water/hydroxide and His 64 found in the SCC-DFTB/MM simulations. These wires were identified following a definition of hydrogen-bond in terms of both distance (O—O < 3.5 A) and angle (O—H—O > 140°) and care... 

Table 7-1. Representative results from statistical analyses used to determine the values and statistical errors of 9 AGch(D)Oh/3 - for the pKa calculation of the zinc-bound water in CAIIa...

Table 7-2. Calculated pKa s of the zinc-bound water in the wild type and E106Q mutant of CAII using thermodynamic integration3...

His residues at configurations sampled using the popular link-host-atom exclusion scheme changes the free energy derivatives by 8-9 kcal/mol despite that the QM/MM frontiers are far from the zinc-bound water. With this effect taken into account, the calculated pKa value for the zinc-bound water in the WT CAII is in encouraging agreement with experiment the value is 7.1 (5.4) for the 20 (25) A-inner-region simulations, as compared to the experimental value of around 7 [86],... 

Before our work [39], only one catalytic mechanism for zinc dependent HDACs has been proposed in the literature, which was originated from the crystallographic study of HDLP [47], a histone-deacetylase-like protein that is widely used as a model for class-I HDACs. In the enzyme active site, the catalytic metal zinc is penta-coordinated by two asp residues, one histidine residues as well as the inhibitor [47], Based on their crystal structures, Finnin et al. [47] postulated a catalytic mechanism for HDACs in which the first reaction step is analogous to the hydroxide mechanism for zinc proteases zinc-bound water is a nucleophile and Zn2+ is five-fold coordinated during the reaction process. However, recent experimental studies by Kapustin et al. suggested that the transition state of HDACs may not be analogous to zinc-proteases [48], which cast some doubts on this mechanism. 

Tris(2-aminoethyl)amine (tren) and hexamethyl tren form pentacoordinate complexes with a bound water. The enthalpies of ionization have been determined.242 These results dispute the data from the systems that led to the view that solvent structure mediates the pAia of zinc bound water in zinc hydrolytic enzymes. The effect was shown to be entirely enthalpic. 

Kimura and co-workers have synthesized a series of alkoxide complexes with the alcohol functionality as a pendent arm.447 674 737 A zinc complex of l-(4-bromophenacyl)-l, 4,7,10-tetraaza-cyclododecane was also synthesized by the same workers to mimic the active site of class II aldolases. The X-ray structure shows a six-coordinate zinc center with five donors from the ligand and a water molecule bound. The ketone is bound with a Zn—O distance of 2.159(3) A (Figure 12). Potentiometric titration indicated formation of a mixture of the hydroxide and the enolate. Enolate formation was also independently carried out by reaction with sodium methoxide, allowing full characterization.738... 

As an example, consider an early calculation of isotope effects on enzyme kinetics by Hwang and Warshel [31]. This study examines isotope effects on the catalytic reaction of carbonic anhydrase. The expected rate-limiting step is a proton transfer reaction from a zinc-bound water molecule to a neighboring water. The TST expression for the rate constant k is... 

Zinc Ion or Zinc-Bound Water as General Acid. 158... 

The observed normal isotope effect of 1.9 provides further evidence supporting the role of Asp55 as the general base. Namely, a normal isotope effect of 1.9 is most consistent with general base catalysis by an amino acid side chain, as inverse isotope effects are commonly observed when a zinc-bound water molecule, or hydroxide, is the attacking nucleophile. For example, the zinc-containing enzymes AMP deaminase [111], thermolysin [112], stromelysin [113], and a desuccinylase [114] are each believed to utilize a zinc-bound water as the nucleophile, and all of these reactions are characterized by an inverse deuterium isotope effect. This inverse isotope effect is thought to result from a dominant... 

The first zinc enzyme to be discovered was carbonic anhydrase in 1940, followed by car-boxypeptidase A some 14 years later. They both represent the archetype of mono-zinc enzymes, with a central catalytically active Zn2+ atom bound to three protein ligands, and the fourth site occupied by a water molecule. Yet, despite the overall similarity of catalytic zinc sites with regard to their common tetrahedral [(XYZ)Zn2+-OH2] structure, these mononuclear zinc enzymes catalyse a wide variety of reactions, as pointed out above. The mechanism of action of the majority of zinc enzymes centres around the zinc-bound water molecule,... 

Figure 12.2 The zinc-bound water can be ionized to zinc-bound hydroxide, polarized by a general base to generate a nucleophile for catalysis or displaced by the substrate. (From McCall et al., 2000. Copyright (2000) the American Society for Nutritional Sciences.)...

The essential features of the catalytic cycle are summarized in Figure 12.6. After binding of NAD+ the water molecule is displaced from the zinc atom by the incoming alcohol substrate. Deprotonation of the coordinated alcohol yields a zinc alkoxide intermediate, which then undergoes hydride transfer to NAD+ to give the zinc-bound aldehyde and NADH. A water molecule then displaces the aldehyde to regenerate the original catalytic zinc centre, and finally NADH is released to complete the catalytic cycle. 

The crystal structure (Strop et al. 2001) reveals a homodimer with the zinc atom ligated by the sulfur atoms of two cysteines (Cys 32 and Cys 90) and the nitrogen atom of a histidine (His 87), as is the case for the plant-type enzyme (Fig. 11.3). The active site contains an HEPES buffer molecule in a position that implicates involvement of Asp 34 in the transport of protons after ionization of the zinc-bound water. 

An example is the hydration of CO2, as catalyzed by carbonic anhydrasek The catalytic reaction requires proton transfer from the zinc-bound water at the active site to solution to regenerate Zn-OH in each catalytic cycle. The most efficient isozyme forms use His-64 as a nearby proton shuttle group other forms contain residues that are less effective in proton transfer and limit overall catalytic efficiency. 

In Cu,Zn-SOD the copper atom is bound to three histidine groups and the zinc is bound to two histidines and an aspartate oxygen atom (Tainer et al., 1982, 1983) (Fig. 34). The Cu"-Zn distance is 6.3 A. The zinc-ligand geometry is tetrahedral, with a strong distortion toward a trigonal pyramid with aspartic acid at the apex. The coordination of the Cu(II) is tetrahedrally distorted square planar. The axial position of copper is more open on the solvent side than on the protein side probably, water is bound there. The Zn(II) is buried, while the Cu(II) site is solvent accessi-... 

Fig. 2. The zinc-bound water molecule of this rigid metalloamide complex exhibits a pKa of about 7 (Groves and Olson, 1985). The complex is a biomimic of the tetracoordinate metal ion in the carboxypeptidase A active site.

Vedani and Huhta (1990) studied the structure of zinc-water interactions retrieved from the Gambridge Structural Database, and a scatter-plot of these interactions is reproduced in Fig. 15. It is obvious that zinc-bound water orients an sp lone electron pair toward the metal ion... 

Fig. 29. The regeneration of zinc-bound hydroxide is achieved by a proton transfer from zinc-bound water through a solvent network to His-64, which then releases a proton to buffer. His-64 may undergo changes in tautomerization and/or conformation during this process.

Zinc proteases carboxypeptidase A and thermolysin have been extensively studied in solution and in the crystal (for reviews, see Matthews, 1988 Christianson and Lipscomb, 1989). Both carboxypeptidase A and thermolysin hydrolyze the amide bond of polypeptide substrates, and each enzyme displays specificity toward substrates with large hydrophobic Pi side chains such as phenylalanine or leucine. The exopeptidase carboxypeptidase A has a molecular weight of about 35K and the structure of the native enzyme has been determined at 1.54 A resolution (Rees et ai, 1983). Residues in the active site which are important for catalysis are Glu-270, Arg-127, (liganded by His-69, His-196, and Glu-72 in bidentate fashion), and the zinc-bound water molecule (Fig. 30). 

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