Enzyme heme group

The most conspicuous use of iron in biological systems is in our blood, where the erythrocytes are filled with the oxygen-binding protein hemoglobin. The red color of blood is due to the iron atom bound to the heme group in hemoglobin. Similar heme-bound iron atoms are present in a number of proteins involved in electron-transfer reactions, notably cytochromes. A chemically more sophisticated use of iron is found in an enzyme, ribo nucleotide reductase, that catalyzes the conversion of ribonucleotides to deoxyribonucleotides, an important step in the synthesis of the building blocks of DNA. 

NO-sensitive GC represents the most important effector enzyme for the signalling molecule NO, which is synthesised by NO synthases in a Ca2+-dependent manner. NO-sensitive GC contains a prosthetic heme group, acting as the acceptor site for NO. Formation of the NO-heme complex leads to a conformational change, resulting in an increase of up to 200-fold in catalytic activity of the enzyme [1]. The organic nitrates (see below) commonly used in the therapy of coronary heart disease exert their effects via the stimulation of this enzyme. 

Besides NO, other sGC-activating substances have been reported Carbon monoxide (CO) is known to bind to heme groups with high affinity but has been shown to activate the enzyme only marginally (three- to fivefold). The compound YC-1 ([3-(5 -hydroxymethyl-2 -fury 1)-1-benzyl indazole]) is a prototype of a new class of so-called NO-sensitisers. YC-1 causes a tenfold activation of NO-sensitive GC. Pharmacologically more interesting, YC-1 increases GC s sensitivity towards NO and CO suggesting potential beneficial effects of... 

The classical peroxidative catalytic cycle involves the formation of a so-called compound I intermediate product of the binding of the hydrogen peroxide to the heme group of the enzyme and the subsequent release of a water molecule. The cycle operates through a second intermediate, compound II, to the resting state enzyme by two individual one-electron withdrawals from the reducing substrates [70],... 

HRP C contains two different types of metal center (i.e., iron(III) protoporphyrin IX-heme group and two calcium atoms) that are fundamental for the integrity of the enzyme. The heme group is attached to the enzyme at His 170 by a coordinate bond between the histidine side-chain NE2 atom and the heme iron atom. The second axial coordination site is unoccupied in the resting state of the enzyme but available to hydrogen peroxide during enzyme turnover. Small molecules such as carbon monoxide, cyanide, fluoride, and azide bind to the heme iron atom at this distal site, giving six-coordinated PX complexes. 

Figure 9.1 CYP catalytic cycle. The sequential two-electron reduction of CYP and the various transient intermediates were first described in the late 1960s [206], The sequence of events that make up the CYP catalytic cycle is shown. The simplified CYP cycle begins with heme iron in the ferric state. In step (i), the substrate (R—H) binds to the enzyme, somewhere nearthe distal region of the heme group and disrupts the water lattice within the enzymes active site [207], The loss of water elicits a change in the heme iron spin state (from low spin to high spin) [208]. Step (ii) involves the transfers of an electron from NADPH via the accessory flavoprotein NADPH-CYP reductase, with the electron flow going from the reductase prosthetic group FAD to FMN to the CYP enzyme [206,209]. The...

There are at least two factors that could influence the turnover rate, the site of metabolism (hot spot) and the affinity of a compound toward these enzymes the protein/ligand (substrate or inhibitor) interaction and the chemical reactivity of the compound towards oxidation. Because of the interaction of the protein with the potential ligand, certain atoms of the compound could be exposed to the heme group, and depending on the chemical nature of these moieties the oxidative reaction will take place at different rates, for example celecoxib is metabolized by CYP2C9 at the... 

Once the protein interaction pattern is translated from Cartesian coordinates into distances from the reactive center of the enzyme and the structure of the ligand has been described with similar fingerprints, both sets of descriptors can be compared [25]. The hydrophobic complementarity, the complementarity of charges and H-bonds for the protein and the substrates are all computed using Carbo similarity indices [26]. The prediction of the site of metabolism (either in CYP or in UGT) is based on the hypothesis that the distance between the reactive center on the protein (iron atom in the heme group or the phosphorous atom in UDP) and the interaction points in the protein cavity (GRID-MIF) should correlate to the distance between the reactive center of the molecule (i.e. positions of hydrogen atoms and heteroatoms) and the position of the different atom types in the molecule [27]. 

The success of Chapman and co-workers in expression of flavocytochrome 2 in E. coli [23] is encouraging in its impUcations for future expression of flavoproteins in this host because, in their experience both the flavin and heme groups are incorporated into the recombinant protein. Moreover, the bacterial expression system produces the protein 500-1000 fold more efficiently than the yeast from which it was cloned. The enzyme produced in E. coli, however, lacks the first five amino acid residues at its amino terminus, a result which presumably reflects subtle differences in protein synthesis between the two organisms. 

Nitric oxide (NO) is synthesized by vascular endothelium in response to vasodilators. It diffuses into the surrounding vascular smooth muscle, where it directly binds the heme group of soluble guanylate cyclase, activating the enzyme. 

Pyridoxal phosphate is a required coenzyme for many enzyme-catalyzed reactions. Most of these reactions are associated with the metabolism of amino acids, including the decarboxylation reactions involved in the synthesis of the neurotransmitters dopamine and serotonin. In addition, pyridoxal phosphate is required for a key step in the synthesis of porphyrins, including the heme group that is an essential player in the transport of molecular oxygen by hemoglobin. Finally, pyridoxal phosphate-dependent reactions link amino acid metabolism to the citric acid cycle (chapter 16). 

The velocities of reactions (1) and (2), and V2, can be expressed in terms of the total enzyme concentration (or total heme groups) [E] and the concentration of enzyme-substrate complex [ES], as... 

Removal of calcium from HRP C has a significant effect not only on enzyme activity and thermal stability, but also on the environment of the heme group. The calcium-depleted enzyme has optical, EPR, and H NMR spectra that are different from those of the native enzyme (211). Temperature dependence studies indicate that the heme iron exists as a thermal admixture of high- and low-spin states. Kinetic measurements at pH 7 show that ki, the rate constant for compound I formation, is only reduced marginally from 1.6 0.1 x 10 to 1.4 x lO M s , whereas k, the rate constant for compound II reduction, is reduced from 8.1 1.6 x 10 to 3.6 x lO M s (reducing substrate p-aminobenzoic acid), 44% of its initial value (211). There can be little doubt that this is the main reason for the loss of enzyme activity on calcium removal. 

The proximal calcium binding site is coupled to the heme group by virtue of the fact that one of its ligands, Thrl71, is adjacent to the proximal histidine residue, Hisl70 (Fig. 4). The results of site-directed mutagenesis studies at this position are awaited with interest. An illustration of the importance of both calcium sites to the structure and function of HRP C is afforded by the need to incorporate calcium as a component of in vitro folding mixtures to obtain active recombinant enzyme from solubilized inclusion bodies (64). 

The relationship between the aromatic donor molecule binding site and the heme group has been explored further using ID and 2D H NMR, and enzyme inactivation studies (see Section H1,D). Nuclear Overhauser enhancement data obtained in ID NOE and 2D NOESY experiments (these provide information on proton-proton distances in a structure, with an upper limit of 5 A) indicate that binding occurs relatively close to heme methyl CI8H3 (229), in agreement with the conclusion from inactivation work that substrates interact with the... 

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