Active clefts

At higher concentrations of Li+, it is proposed that the observed noncompetitive inhibition of IMPase [105] is due to Li+ binding to the first Mg2+ site on the free enzyme [115]. As predicted from the crystal structure, this site is located deep within the active cleft of the enzyme and is therefore relatively inaccessible to the Li+. 

Bovine a-lactalbumin is one of the two enzymes in lactose synthetase, and its amino acid sequence shows striking similarities to that of lysozyme.118 A model based on the lysozyme model has been built, and the side-chain interactions found are convincing, showing that the model is essentially correct. The active cleft in the crystal is, however, shorter than that in the model, and is consistent with a mono- or di-saccharide as the substrate. Thus, the lysozyme structure may serve as a model for some enzymes that synthesize and hydrolyze carbohydrates. 

Several extracellular enzymes have one or more Ca + ions as integral parts of their structure. In a very few of them the Ca ion is bound at or near the active cleft, and appears necessary for maintaining the catalytic activity (phospholipase A2, a-amylase, nucleases), whereas other enzymes show catalytic activity even in the absence of Ca (trypsin and other serine proteases). In the latter proteins, the Ca + ion is usually ascribed a structural role, although its function may be rather more related to dynamics and so be more subtle and complex. 

In a study of the extension of the transition phenomenon in hen egg-white lysozyme crystals the case of monoclinic rather than tetragonal crystals as the starting materials was considered. The rotational and transitional parameters relating to the known co-ordinates of the crystalline form of hen egg-white lysozyme molecule have been related to those of the turkey enzyme which differs in its primary structure in 7 of the 129 residues. An electron density map has been calculated at 5 A resolution the packing of the molecules in this form appears to present the entire length of the active cleft in the vicinity of the crystallographic six-fold axis and does not appear to be blocked by neighbouring molecules. 

In spite of the fact that the X-ray structure of carbonic anhydrase is known to a resolution of about 2 A the detailed mechanism of the enzyme is still not known (66). Ward (67) studied the Cl NMR linewidth on a 0.5 M NaCl solution containing about 4 X 10 M bovine carbonic anhydrase. The observed excess line broadening was attributed to a coordination of Cl" to the zinc atom in the active cleft. Titration with acetazolamide, i hich is a strong inhibitor of the enzyme, and CN " showed that these ligands almost entirely eliminated the excess line broadening. The results from the acetazolamide titration are shown in Figure 6. An equivalence point is obtained at a 1 1 molar ratio... 

The conclusion of Ward that Cl binds directly to the Zn at the active cleft has been questioned by Koenig and Brown (70). From a comparison of water proton and Cl relaxation data they argued that competition between Cl and water for a common Zn site, as also implied in Ward s model, does not occur. They Instead suggest that Cl may be bound to a cationic residue elsewhere on the protein. Nome et al. have reported that the Cl excess linewidth observed in carbonic anhydrase solutions is markedly affected by the addition of the anion Au(CN)2 (71). This anion binds very poorly to Zn and other metal ions. This result also indicates that Cl- is not directly coordinated to zinc. 

Dopamine. Dopamine (DA) (2) is an intermediate in the synthesis of NE and Epi from tyrosine. DA is localized to the basal ganglia of the brain and is involved in the regulation of motor activity and pituitary hormone release. The actions of DA are terminated by conversion to dihydroxyphenylacetic acid (DOPAC) by monoamine oxidase-A and -B (MAO-A and -B) in the neuron following reuptake, or conversion to homovanillic acid (HVA) through the sequential actions of catechol-0-methyl transferase (COMT) and MAO-A and -B in the synaptic cleft. 

Another class of DNA-binding proteins are the polymerases. These have a nonspecific interaction with DNA because the same protein acts on all DNA sequences. DNA polymerase performs the dual function of DNA repHcation, in which nucleotides are added to a growing strand of DNA, and acts as a nuclease to remove mismatched nucleotides. The domain that performs the nuclease activity has an a/P-stmcture, a deep cleft that can accommodate double-stranded DNA, and a positively charged surface complementary to the phosphate groups of DNA. The smaller domain contains the exonuclease active site at a smaller cleft on the surface which can accommodate a single nucleotide. 

P-Lactam antibiotics exert their antibacterial effects via acylation of a serine residue at the active site of the bacterial transpeptidases. Critical to this mechanism of action is a reactive P-lactam ring having a proximate anionic charge that is necessary for positioning the ring within the substrate binding cleft (24). 

In free CDK2 the active site cleft is blocked by the T-loop and Thr 160 is buried (Figure 6.20a). Substrates cannot bind and Thr 160 cannot be phosphorylated consequently free CDK2 is inactive. The conformational changes induced by cyclin A binding not only expose the active site cleft so that ATP and protein substrates can bind but also rearrange essential active site residues to make the enzyme catalytically competent (Figure 6.20b). In addition Thr... 

Figure 6.20 Space-filling diagram illustrating the structural changes of CDK2 upon cyclin binding, (a) The active site is in a cleft between the N-terminal domain (blue) and the C-terminal domain (purple). In the inactive form this site is blocked by the T-loop.

Figure 16.21 Structure of one subunit of the core protein of Slndbls virus. The protein has a similar fold to chymotrypsin and other serine proteases, comprising two Greek key motifs separated by an active site cleft. The C-terminus of the protein is bound in the catalytic site, making the coat protein inactive (Adapted from S. Lee et al., Structure 4 531-541, 1996.)...

Each precursor protein molecule is cleaved only once to generate one molecule of the coat protein, and catalytic activity is restricted to the precursor protein. Why is the coat protein itself catalytically inactive The structure of the coat protein shows that its C-terminus is bound in the active site cleft and thereby prevents other proteins entering the cleft and being cleaved. Tbis arrangement allows the precursor protein to fulfill its function to generate the coat protein and prevents the coat protein from destroying other proteins in the infected cell, including other coat proteins. 

Citrate synthase in mammals is a dimer of 49-kD subunits (Table 20.1). On each subunit, oxaloacetate and acetyl-CoA bind to the active site, which lies in a cleft between two domains and is surrounded mainly by a-helical segments (Figure 20.6). Binding of oxaloacetate induces a conformational change that facilitates the binding of acetyl-CoA and closes the active site, so that the reactive carbanion of acetyl-CoA is protected from protonation by water. 

Figure 5.9 Models of hexo-kinase in space-filling and wireframe formats, showing the cleft that contains the active site where substrate binding and reaction catalysis occur. At the bottom is an X-ray crystal structure of the enzyme active site, showing the positions of both glucose and ADP as well as a lysine amino acid that acts as a base to deprotonate glucose.

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