Histidine intermediates

Analysis of deacylation by histidinyl-functionalized micelles suggests that the histidinyl group can act both nucleophilically, generating an acylated histidine intermediate, and as a general base. These conclusions are consistent with the kinetic solvent hydrogen isotope effect (Murakami et al., 1981). 

In the resting (oxidized) state (1) the iron atoms of the binuclear fes-Fes site are antiferromagnetically coupled via a single strong Fe-O-Fe bond. The heme bs iron is five-coordinate in the oxidized state (1) upon reduction (2) the heme Fe is still five-coordinate, but bound to the proximal histidine. Intermediate (2) is ready to bind the two molecules of NO. Kmnita et a/. attempt to answer the question whether both NO molecules bind to Fes (the cis mechanism dzs or one to bs, the other to Fes as depicted in (3) of Figure 5(a) (the trans mechanism " ). 

The products suggest activation as a phosphoryl rather than as a nucleotidyl derivative. Both succinyl phosphate and thiophosphoryl coenzyme A have been suggested as intermediates. However, neither is included, at least as a freely dissociable intermediate, in current formulations of this reaction. An enzyme-bound phosphoryl histidine intermediate is thought to be involved, as is some sort of activated enzyme-CoA complex. Many aspects of the enzyme mechanism are still in doubt, but the sequence below is consistent with most available data. 

The results supported the proposal of Glu-165 as the general base and suggested the novel possibility of neutral histidine acting as an acid, contrary to the expectation that His-95 was protonated [26,58]. The conclusion that the catalytic His-95 is neutral has been confinned by NMR spectroscopy [60]. The selection of neutral imidazole as the general acid catalyst has been discussed in terms of achieving a pX, balance with the weakly acidic intermediate. This avoids the thermodynamic trap that would result from a too stable enediol intermediate, produced by reaction with the more acidic imidazolium [58]. 

Certain amino acids and their derivatives, although not found in proteins, nonetheless are biochemically important. A few of the more notable examples are shown in Figure 4.5. y-Aminobutyric acid, or GABA, is produced by the decarboxylation of glutamic acid and is a potent neurotransmitter. Histamine, which is synthesized by decarboxylation of histidine, and serotonin, which is derived from tryptophan, similarly function as neurotransmitters and regulators. /3-Alanine is found in nature in the peptides carnosine and anserine and is a component of pantothenic acid (a vitamin), which is a part of coenzyme A. Epinephrine (also known as adrenaline), derived from tyrosine, is an important hormone. Penicillamine is a constituent of the penicillin antibiotics. Ornithine, betaine, homocysteine, and homoserine are important metabolic intermediates. Citrulline is the immediate precursor of arginine. 

The mechanism of succinyl-CoA synthetase is postulated to involve displacement of CoA by phosphate, forming succinyl phosphate at the active site, followed by transfer of the phosphoryl group to an active-site histidine (making a phosphohistidine intermediate) and release of succinate. The phosphoryl moiety is then transferred to GDP to form GTP (Figure 20.13). This sequence of steps preserves the energy of the thioester bond of succinyl-CoA in a series of high-energy intermediates that lead to a molecule of ATP ... 

The tetrahedral intermediate expels a diacylglycerol as the leaving group and produces an acyl enzyme. The step is catalyzed by a proton transfer from histidine to make the leaving group a neutral alcohol. 

Q The enzyme active site contains an aspartic acid, a histidine, and a serine. First, histidine acts as a base to deprotonate the -OH group of serine, with the negatively charged carboxylate of aspartic acid stabilizing the nearby histidine cation that results. Serine then adds to the carbonyl group of the triacylglycerol, yielding a tetrahedral intermediate. 

This intermediate expels a dtacylglycerol as leaving group in a nucleophilic acyl substitution reaction, giving an acyl enzyme. The dtacylglycerol is protonated by the histidine cation. 

The tetrahedral intermediate expels the serine as leaving group in a second nucleophilic acyl substitution reaction, yielding a free fatty acid. The serine accepts a proton from histidine, and the enzyme has now returned to its starting structure. 

Interestingly, however, the mechanisms of the two phosphate hydrolysis reactions in steps 9 and 11 are not the same. In step 9, water is the nucleophile, but in the glucose 6-phosphate reaction of step 11, a histidine residue on the enzyme attacks phosphorus, giving a phosphoryl enzyme intermediate that subsequently reacts with water. 

Much less is known about the participation of sugars in the biosynthesis of pyramine in yeasts, and although it has been proven that sugars can provide some carbon atoms, the exact nature of the more advanced intermediates of sugar origin is not yet clear. Some features of the biosynthesis in S. cerevisiae are summarized in Scheme 33. Two l5N atoms from DL-(l,3-,5N2)histidine were incorporated into the N-3 and amino nitrogen atoms of pyramine. The nitrogen atom of (,5N)aspartate, a known precursor of N-l of histidine, was incorporated into pyramine without dilution.58-70 It was also found that N-l and C-2 of pyramine came respectively from N-l and C-2 of pyridoxol.71-73... 

The mechanism for the lipase-catalyzed reaction of an acid derivative with a nucleophile (alcohol, amine, or thiol) is known as a serine hydrolase mechanism (Scheme 7.2). The active site of the enzyme is constituted by a catalytic triad (serine, aspartic, and histidine residues). The serine residue accepts the acyl group of the ester, leading to an acyl-enzyme activated intermediate. This acyl-enzyme intermediate reacts with the nucleophile, an amine or ammonia in this case, to yield the final amide product and leading to the free biocatalyst, which can enter again into the catalytic cycle. A histidine residue, activated by an aspartate side chain, is responsible for the proton transference necessary for the catalysis. Another important factor is that the oxyanion hole, formed by different residues, is able to stabilize the negatively charged oxygen present in both the transition state and the tetrahedral intermediate. 

In vanadium-dependent haloperoxidases, the metal center is coordinated to the imidazole system of a histidine residue, which is similarly responsible for creating hypochlorite or hypobromite as electrophilic halogenating species [274]. Remarkably, a representative of this enzyme class is capable of performing stereoselective incorporation of halides, as has been reported for the conversion of nerolidol to various snyderols. The overall reaction commences through a bromonium intermediate, which cyclizes in an intramolecular process the resulting carbocation can ultimately be trapped upon elimination to three snyderols (Scheme 9.37) [275]. 

The rate of phosphoprotein formation in the presence of 5 mM CaCl2 was only slightly affected by mild photooxidation in the presence of Rose Bengal, but the hydrolysis of phosphoenzyme intermediate was inhibited sufficiently to account for the inhibition of ATP hydrolysis [359]. The extent of inhibition was similar whether the turnover of E P was followed after chelation of Ca with EGTA, or after the addition of large excess of unlabeled ATP. These observations point to the participation of functionally important histidine residues in the hydrolysis of phosphoprotein intermediate [359]. 

Similarly, the rate of inhibition of phosphoenzyme formation by diethylpyrocarbonate (DEPC) was much slower than the loss of ATPase activity [368], Even when the reaction approached completion with more than 90% inhibition of ATP hydrolysis, about 70% of the Ca -ATPase could still be phosphorylated by ATP (2.3nmoles of E P/mg protein). The remaining 30% of E P formation and the corresponding ATPase activity was not reactivated by hydroxylamine treatment, suggesting some side reaction with other amino acids, presumably lysine. When the reaction of the DEPC-modified ATPase with P-ATP was quenched by histidine buffer (pH 7.8) the P-phosphoenzyme was found to be exceptionally stable under the same conditions where the phosphoenzyme formed by the native ATPase underwent rapid hydrolysis [368]. The nearly normal phosphorylation of the DEPC-trea-ted enzyme by P-ATP implies that the ATP binding site is not affected by the modification, and the inhibition of ATPase activity is due to inhibition of the hydrolysis of the phosphoenzyme intermediate [368]. This is in contrast to an earlier report by Tenu et al. [367], that attributed the inhibition of ATPase activity by... 

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