Active site residue

The -amino group of lysine-87 forms external aldimine E in Fig. 7.6 and is released after L-serine binds to form ES I in Fig. 7.6 Lysine-87 is ideally situated to facilitate catalysis by removing the a-proton of L-serine.7 We find that a mutant form of the a2Pi complex in which the p subunit lysine-87 is replaced by threonine (K87T) is inactive but binds L-serine and L-tryptophan slowly and extremely tightly.92-97 These results provide evidence that 

An important intermediate step in the reaction of L-serine and indole to form l-tryptophan is the nucleophilic attack of indole on the Schiff base of amino acrylate (ES III 

The transition state for the enzymatic reaction has been shown to have a chairlike geometry as well [61], and conformationally constrained compounds that mimic this structure, such as the oxabicyclic dicarboxylic acid 1 (Fig. 3.6), are good inhibitors of chorismate mutase enzymes [62 - 64], How a protein might stabilize this high-energy species has been a matter of some debate. Recently, heavy atom isotope effects were used to characterize the structure of the transition state bound to BsCM [65]. A very 

The results of these selection experiments are mechanistically significant insofar as they support a critical role for a cation in the mechanism of chorismate mutase. No active catalysts were found that lacked a cation in the vicinity of the substrate s ether oxygen. The conclusion that a cation is crucial for high chorismate mutase activity can thus be made with much greater confidence than would have been possible following a conventional mutagenesis experiment in which only single substitutions were considered [66]. Of course, we cannot exclude the possibility that other, equally effective 

Evolutionary Strategies to Investigate the Structure and Function of Chorismate Mutases 

The Fab fragment of 1F7 has already been shown to function in the cytoplasm of a chorismate mutase-deficient yeast strain [43,44]. When produced at a sufficiently high level, the catalytic antibody is able to replace the missing enzyme and weakly complement the metabolic defect. Conceivably, therefore, it can be placed under selection pressure to identify variants that have higher catalytic efficiency. Preliminary results from such experiments appear quite promising [69]. 

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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 6.18 The PSTAIRE helix undergoes a major conformational change when CDK2 binds to cyclin A. In the inactive free CDK2 (yellow) the active site residue Glu 51 is far from the active site. Upon binding of cyclin A to CDK2 the PSTAIRE helix (hiue) rotates 90° and changes its position so that Glu 51 becomes positioned into the active site. (Adapted from P.D. Jeffry et al.. Nature 376 313-320, 1995.)...

Figure 11.10 Topological diagram of the two domains of chymotrypsin, illustrating that the essential active-site residues are part of the same two loop regions (3-4 and 5-6, red) of the two domains. These residues form the catalytic triad, the oxyanion hole (green), and the substrate binding regions (yellow and blue) including essential residues in the specificity pocket.

From a map at low resolution (5 A or higher) one can obtain the shape of the molecule and sometimes identify a-helical regions as rods of electron density. At medium resolution (around 3 A) it is usually possible to trace the path of the polypeptide chain and to fit a known amino acid sequence into the map. At this resolution it should be possible to distinguish the density of an alanine side chain from that of a leucine, whereas at 4 A resolution there is little side chain detail. Gross features of functionally important aspects of a structure usually can be deduced at 3 A resolution, including the identification of active-site residues. At 2 A resolution details are sufficiently well resolved in the map to decide between a leucine and an isoleucine side chain, and at 1 A resolution one sees atoms as discrete balls of density. However, the structures of only a few small proteins have been determined to such high resolution. 

FIGURE 16.16 Comparison of the amino acid sequences of chymotrypsinogen, trypsino-gen, and elastase. Each circle represents one amino acid. Nmnbering is based on the sequence of chymotrypsinogen. Filled circles indicate residues that are identical in all three proteins. Disnlfide bonds are indicated in yellow. The positions of the three catalytically important active-site residues (His, Asp °-, and Ser ) are indicated. 

Definitive identification of lysine as the modified active-site residue has come from radioisotope-labeling studies. NaBH4 reduction of the aldolase Schiff base intermediate formed from C-labeled dihydroxyacetone-P yields an enzyme covalently labeled with C. Acid hydrolysis of the inactivated enzyme liberates a novel C-labeled amino acid, N -dihydroxypropyl-L-lysine. This is the product anticipated from reduction of the Schiff base formed between a lysine residue and the C-labeled dihydroxy-acetone-P. (The phosphate group is lost during acid hydrolysis of the inactivated enzyme.) The use of C labeling in a case such as this facilitates the separation and identification of the telltale amino acid. 

A different mechanism operates in the wheat germ enzyme. 2,3-Bisphosphoglycerate is not a cofactor. Instead, the enzyme carries out intra-molecular phosphoryl group transfer (Figure 19.25). The C-3 phosphate is transferred to an active-site residue and then to the C-2 position of the original substrate molecule to form the product, 2-phosphoglycerate. 

Determining active site residues, and residues specific for subfamilies... 

Threonine peptidases (and some cysteine and serine peptidases) have only one active site residue, which is the N-terminus of the mature protein. Such a peptidase is known as an N-terminal nucleophile hydrolase or Ntn-hydrolase. The amino group of the N-terminal residue performs the role of the general base. The catalytic subunits of the proteasome are examples of Ntn-hydrolases. 

The order and nature of the active site residues and metal ligands is conserved between homologous peptidases. All the members of a family will have the same catalytic type. 

Peptidases have been classified by the MEROPS system since 1993 [2], which has been available viatheMEROPS database since 1996 [3]. The classification is based on sequence and structural similarities. Because peptidases are often multidomain proteins, only the domain directly involved in catalysis, and which beais the active site residues, is used in comparisons. This domain is known as the peptidase unit. Peptidases with statistically significant peptidase unit sequence similarities are included in the same family. To date 186 families of peptidase have been detected. Examples from 86 of these families are known in humans. A family is named from a letter representing the catalytic type ( A for aspartic, G for glutamic, M for metallo, C for cysteine, S for serine and T for threonine) plus a number. Examples of family names are shown in Table 1. There are 53 families of metallopeptidases (24 in human), 14 of aspartic peptidases (three of which are found in human), 62 of cysteine peptidases (19 in human), 42 of serine peptidases (17 in human), four of threonine peptidases (three in human), one of ghitamicpeptidases and nine families for which the catalytic type is unknown (one in human). It should be noted that within a family not all of the members will be peptidases. Usually non-peptidase homologues are a minority and can be easily detected because not all of the active site residues are conserved. 

All peptidases within a family will have a similar tertiary structure, and it is not uncommon for peptidases in one family to have a similar structure to peptidases in another family, even though there is no significant sequence similarity. Families of peptidases with similar structures and the same order of active site residues are included in the same clan. A clan name consists of two letters, the first representing the catalytic type as before, but with the extra letter P , and the second assigned sequentially. Unlike families, a clan may contain peptidases of more than one catalytic type. So far this has only been seen for peptidases with protein nucleophiles, and these clans are named with an initial P . Only three such clans are known. Clan PA includes peptidases with a chymotrypsin-like fold, which besides serine peptidases such as chymotrypsin... 

Similar reaction mechanisms, involving general base and metal ion catalysis, in conjunction with an OH nucleophilic attack, have been proposed for thermolysin (Ref. 12) and carboxypeptidase A (Refs. 12 and 13). Both these enzymes use Zn2+ as their catalytic metal and they also have additional positively charged active site residues (His 231 in thermolysin and... 

Fig. 3 Sialidase inhibitor Neu5Ac2en 4 bound in the active site of influenza A virus sialidase (from PDB structure IfSb (Smith et al, 2001)), Left Stick model of 4 surrounded by some important active site residues. Right Electrostatic potential surface rendering of the active site (blue -positive, red - negative), (Amino acid numbering for influenza A/N2 sialidase is used throughout this review)...

Fig. 6 Superimposition of inhibitors and key active site residues from crystal structures of oseltamivir carboxylate 18 brown carbons, PDB - 2qwk) and Neu5Ac2en 4 (green carbons, PDB - IfSb) in complex with influenza A virus siaMdase. Note the alternative conformations of the...

Importantly, the crystal structure of 34 complexed with N9 sialidase (Fig. 8) indicated differences in the orientation of the guanidino group in subsite S2, and in its interaction with the active site residues, compared to that of zanamivir (Babu et al. 2000). These differences have implications for cross-reactivity of 34 with zanamivir-resistant influenza viruses that have Glul 19 mutations in the sialidase S2 subsite (see Sect. 5.1). 

Lewis DFV, Eddershaw PJ, Goldfarb PS, Tarbit MH. Molecular modelling of CYP3A4 from an alignment with CYP102 identification of key interactions between putative active site residues and CYP3A- specific chemicals. Xenobiotica 1996 10 1067-86. 

Fructose-2,6-bisphosphatase, a regulatory enzyme of gluconeogenesis (Chapter 19), catalyzes the hydrolytic release of the phosphate on carbon 2 of fructose 2,6-bisphosphate. Figure 7-8 illustrates the roles of seven active site residues. Catalysis involves a catalytic triad of one Glu and two His residues and a covalent phos-phohistidyl intermediate. 

Selenocysteine, an essential active site residue in several mammahan enzymes, arises by co-translational insertion of a previously modified tRNA. 

The crystal structure of the HNL isolated from S. bicolor (SbHNL) was determined in a complex with the inhibitor benzoic acid." The folding pattern of SbHNL is similar to that of wheat serine carboxypeptidase (CP-WII)" and alcohol dehydrogenase." A unique two-amino acid deletion in SbHNL, however, is forcing the putative active site residues away from the hydrolase binding site toward a small hydrophobic cleft, thereby defining a completely different active site architecture where the triad of a carboxypeptidase is missing. 

Figure 1 Structure of selected active site residues of MeHNL complexed with...

This study proposes amino acids most likely involved in the Ca -binding site, two putative distinct active sites, residues that probably fold in parallel behx or constitute the Asn ladder. 

However, diffusion of the reactive QM out of the enzyme active site is a major concern. For instance, a 2-acyloxy-5-nitrobenzylchloride does not modify any nucleophilic residue located within the enzyme active site but becomes attached to a tryptophan residue proximal to the active site of chymotrypsin or papain.23,24 The lack of inactivation could also be due to other factors the unmasked QM being poorly electrophilic, active site residues not being nucleophilic enough, or the covalent adduct being unstable. Cyclized acyloxybenzyl molecules of type a could well overcome the diffusion problem. They will retain both the electrophilic hydroxybenzyl species b, and then the tethered QM, in the active site throughout the lifetime of the acyl-enzyme (Scheme 11.1). This reasoning led us to synthesize functionalized... 

To date, 15 GPI variants have been analyzed at the molecular level, and 16 mis-sense mutations, 1 nonsense mutation, and 1 splicing mutation due to a four-nucleotide deletion have been reported (Fig. 7) (B9, F13, K14, Wl, XI). The GPI gene mutations were heterogeneous, although most GPI variants had common biochemical characteristics such as heat instability and normal kinetic properties. We have determined the molecular abnormalities of four homozygous variants, GPI Matsumoto, GPI Iwate, GPI Narita, and GPI Fukuoka (K14). GPI Narita has a homozygous mutation from A to G at position 1028 (343 Gin to Arg), and the same mutation was reported in an Italian patient, GPI Moscone (B9). The substituted Gin is adjacent to the reported active site residue, 341 Asp. Homozygous missense mutations, C to T at position 14 (5 Thr to lie) and C to T at position 671 (224 Thr to Met) have been identified in GPI Matsumoto and GPI Iwate, respectively. GPI... 

However, the active site is only a conceptual tool and the assignment of the active-site atoms is more or less arbitrary. It is not possible to know beforehand which residues and protein interactions that will turn out to be important for the studied reaction. Hybrid QM/MM methods have been used to extend the active site only models by incorporating larger parts of the protein matrix in studies of enzymatic reactions [19-22], The problem to select active-site residues appears both for active-site and QM/MM models, but in the latter, explicit effects of the surrounding protein (i.e. atoms outside the active-site selection) can at least be approximately evaluated. As this and several other contributions in this volume show, this is in many cases highly desirable. 

One reason for the relatively large RMS deviations, compared to the active sites of MMO and RNR, is that the active-site residues are not coordinated to the selenium (see Figure 2-8). The lack of a structural anchor leads to a relatively unstable active-site geometry. An alternative formulation is that the presence of a metal center with strong ligand interactions is one reason the active-site model works comparatively well for many metal enzymes. 

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