The geometry of interactions between catalytic residues and their substrates

The process of deducing the catalytic mechanism of an enzyme from its structure is highly complex and requires extensive experimental work to validate a proposed mechanism. As one step towards improving the reliability of this process, we have gathered statistics describing the typical geometry of catalytic residues with regard to the substrate and one another. In order to analyse residue-substrate interactions, we have assembled a dataset of structures of enzymes of known mechanism bound to substrate, product, or a substrate analogue. Despite the challenges presented in obtaining such experimental data, we were able to include 42 enzyme structures. We have also assembled a separate dataset of catalytic residues which act upon other catalytic residues, using a set of 60 enzyme structures. For both datasets, we have extracted the distances between residues with a given catalytic function and their target moieties. The geometry of residues whose function involves the transfer or sharing of hydrogens (either with substrate or another residue) was analysed more closely. The results showed that the geometry for such productive interactions (prior to the transition state) closely resembles that seen in non-catalytic hydrogen bonds, with distances and angles in the normal expected range. Such statistics provide limits on "expected geometries" for catalytic residues, which will help to identify these residues and elucidate enzyme mechanisms.     

SABER: a computational method for identifying active sites for new reactions

A software suite, SABER (Selection of Active/Binding sites for Enzyme Redesign), has been developed for the analysis of atomic geometries in protein structures, using a geometric hashing algorithm (Barker and Thornton, Bioinformatics 2003;19:1644–1649). SABER is used to explore the Protein Data Bank (PDB) to locate proteins with a specific 3D arrangement of catalytic groups to identify active sites that might be redesigned to catalyze new reactions. As a proof‐of‐principle test, SABER was used to identify enzymes that have the same catalytic group arrangement present in o‐succinyl benzoate synthase (OSBS). Among the highest‐scoring scaffolds identified by the SABER search for enzymes with the same catalytic group arrangement as OSBS were L ‐Ala D/L ‐Glu epimerase (AEE) and muconate lactonizing enzyme II (MLE), both of which have been redesigned to become effective OSBS catalysts, demonstrated by experiments. Next, we used SABER to search for naturally existing active sites in the PDB with catalytic groups similar to those present in the designed Kemp elimination enzyme KE07. From over 2000 geometric matches to the KE07 active site, SABER identified 23 matches that corresponded to residues from known active sites. The best of these matches, with a 0.28 Å catalytic atom RMSD to KE07, was then redesigned to be compatible with the Kemp elimination using RosettaDesign. We also used SABER to search for potential Kemp eliminases using a theozyme predicted to provide a greater rate acceleration than the active site of KE07, and used Rosetta to create a design based on the proteins identified.     

Analysis of catalytic residues in enzyme active sites

We present an analysis of the residues directly involved in catalysis in 178 enzyme active sites. Specific criteria were derived to define a catalytic residue, and used to create a catalytic residue dataset, which was then analysed in terms of properties including secondary structure, solvent accessibility, flexibility, conservation, quaternary structure and function. The results indicate the dominance of a small set of amino acid residues in catalysis and give a picture of a general active site environment. It is hoped that this information will provide a better understanding of the molecular mechanisms involved in catalysis and a heuristic basis for predicting catalytic residues in enzymes of unknown function.     

Computational evidence for the catalytic mechanism of glutaminyl cyclase. A DFT investigation

The results of a DFT theoretical investigation on the catalytic mechanism of the QC enzyme are presented. A rather large model-system is used. It includes the most important residues that are believed to play a key-role in the catalysis. The computational results show that the rate-determining step of the catalytic process is not the nucleophilic attack leading to the cycle formation (a very easy and fast process with a negligible barrier of 0.8 kcal mol(-1)), but a proton transfer, which is assisted by the Glu201 residue acting as a proton shuttle (general base and general acid). A complex network of hydrogen bonds (involving Asp248 and other residues) contribute to lower the activation barrier for the proton shift which affords the formation of an ammonia molecule bonded to the substrate. The ammonia molecule is a good leaving group which is easily expelled from the substrate in the last step of the catalytic cycle, but remains anchored to the enzyme as a ligand of the zinc cation. The metal plays a key-role in assisting the nucleophilic attack (electrostatic catalysis) since it polarizes the substrate gamma-amide carbonyl group (its electrophilic character increases). Also, the strength of the nucleophilic nitrogen (substrate alpha-amino group) is enhanced by hydrogen bonds involving the Glu201 residue. The computations outline the important role of Trp329 in helping the substrate binding process and stabilizing the cyclization transition state.     

Using a library of structural templates to recognise catalytic sites and explore their evolution in homologous families

Catalytic site structure is normally highly conserved between distantly related enzymes. As a consequence, templates representing catalytic sites have the potential to succeed at function prediction in cases where methods based on sequence or overall structure fail. There are many methods for searching protein structures for matches to structural templates, but few validated template libraries to use with these methods. We present a library of structural templates representing catalytic sites, based on information from the scientific literature. Furthermore, we analyse homologous template families to discover the diversity within families and the utility of templates for active site recognition. Templates representing the catalytic sites of homologous proteins mostly differ by less than 1A root mean square deviation, even when the sequence similarity between the two proteins is low. Within these sets of homologues there is usually no discernible relationship between catalytic site structure similarity and sequence similarity. Because of this structural conservation of catalytic sites, the templates can discriminate between matches to related proteins and random matches with over 85% sensitivity and predictive accuracy. Templates based on protein backbone positions are more discriminating than those based on side-chain atoms. These analyses show encouraging prospects for prediction of functional sites in structural genomics structures of unknown function, and will be of use in analyses of convergent evolution and exploring relationships between active site geometry and chemistry. The template library can be queried via a web server at and is available for download.     

Looking at enzymes from the inside out: the proximity of catalytic residues to the molecular centroid can be used for detection of active sites and enzyme-ligand interfaces

Analysis of the distances of the exposed residues in 175 enzymes from the centroids of the molecules indicates that catalytic residues are very often found among the 5% of residues closest to the enzyme centroid. This property of catalytic residues is implemented in a new prediction algorithm (named EnSite) for locating the active sites of enzymes and in a new scheme for re-ranking enzyme-ligand docking solutions. EnSite examines only 5% of the molecular surface (represented by surface dots) that is closest to the centroid, identifying continuous surface segments and ranking them by their area size. EnSite ranks the correct prediction 1-4 in 97% of the cases in a dataset of 65 monomeric enzymes (rank 1 for 89% of the cases) and in 86% of the cases in a dataset of 176 monomeric and multimeric enzymes from all six top-level enzyme classifications (rank 1 in 74% of the cases). Importantly, identification of buried or flat active sites is straightforward because EnSite "looks" at the molecular surface from the inside out. Detailed examination of the results indicates that the proximity of the catalytic residues to the centroid is a property of the functional unit, defined as the assembly of domains or chains that form the active site (in most cases the functional unit corresponds to a single whole polypeptide chain). Using the functional unit in the prediction further improves the results. The new property of active sites is also used for re-evaluating enzyme-inhibitor unbound docking results. Sorting the docking solutions by the distance of the interface to the centroid of the enzyme improves remarkably the ranks of nearly correct solutions compared to ranks based on geometric-electrostatic-hydrophobic complementarity scores.     

Reaction mechanism of azoreductases suggests convergent evolution with quinone oxidoreductases

Azoreductases are involved in the bioremediation by bacteria of azo dyes found in waste water. In the gut flora, they activate azo pro-drugs, which are used for treatment of inflammatory bowel disease, releasing the active component 5-aminosalycilic acid. The bacterium P. aeruginosa has three azoreductase genes, paAzoR1, paAzoR2 and paAzoR3, which as recombinant enzymes have been shown to have different substrate specificities. The mechanism of azoreduction relies upon tautomerisation of the substrate to the hydrazone form. We report here the characterization of the P. aeruginosa azoreductase enzymes, including determining their thermostability, cofactor preference and kinetic constants against a range of their favoured substrates. The expression levels of these enzymes during growth of P. aeruginosa are altered by the presence of azo substrates. It is shown that enzymes that were originally described as azoreductases, are likely to act as NADH quinone oxidoreductases. The low sequence identities observed among NAD(P)H quinone oxidoreductase and azoreductase enzymes suggests convergent evolution.     

氢酶结构及催化机理研究进展

氢酶是一类催化氢的氧化或质子还原的酶,它在微生物产氢过程中扮演着重要角色。根据氢酶所含的金属元素,可分为NiFe_氢酶、Fe-氢酶和不含金属元素的metal_free氢酶。大多数氢酶含有金属原子,它们参与氢酶活性中心和[Fe_S]簇的形成。氢酶的活性中心直接催化氢的氧化与质子的还原,[Fe_S]簇则参与氢酶催化过程中电子的传输。目前已有数种NiFe_氢酶和Fe_氢酶的X射线衍射晶体结构被阐明。根据metal_free氢酶的序列特征,推断其结构与NiFe_氢酶和Fe_氢酶之间存在较大差异。对氢酶活性中心和[Fe_S]簇的深入研究,揭示了氢酶催化反应的机理。     

Capturing the geometry,function, and evolution of enzymes with 3D templates

Structural templates are 3D signatures representing protein functional sites, such as ligand binding cavities, metal coordination motifs, or catalytic sites. Here we explore methods to generate template libraries and algorithms to query structures for conserved 3D motifs. Applications of templates are discussed, as well as some exemplar cases for examining evolutionary links in enzymes. We also introduce the concept of using more than one template per structure to represent flexible sites, as an approach to better understand catalysis through snapshots captured in enzyme structures. Functional annotation from structure is an important topic that has recently resurfaced due to the new more accurate methods of protein structure prediction. Therefore, we anticipate that template‐based functional site detection will be a powerful tool in the task of characterizing a vast number of new protein models.     

The mechanism of aconitase: 1.8 A resolution crystal structure of the S642a:citrate complex

The crystal structure of the S642A mutant of mitochondrial aconitase (mAc) with citrate bound has been determined at 1.8 A resolution and 100 K to capture this binding mode of substrates to the native enzyme. The 2.0 A resolution, 100 K crystal structure of the S642A mutant with isocitrate binding provides a control, showing that the Ser --> Ala replacement does not alter the binding of substrates in the active site. The aconitase mechanism requires that the intermediate product, cis-aconitate, flip over by 180 degrees about the C alpha-C beta double bond. Only one of these two alternative modes of binding, that of the isocitrate mode, has been previously visualized. Now, however, the structure revealing the citrate mode of binding provides direct support for the proposed enzyme mechanism.     

The crystal structure of a bacterial class II ketol-acid reductoisomerase: domain conservation and evolution

Ketol-acid reductoisomerase (KARI; EC 1.1.1.86) catalyzes two steps in the biosynthesis of branched-chain amino acids. Amino acid sequence comparisons across species reveal that there are two types of this enzyme: a short form (Class I) found in fungi and most bacteria, and a long form (Class II) typical of plants. Crystal structures of each have been reported previously. However, some bacteria such as Escherichia coli possess a long form, where the amino acid sequence differs appreciably from that found in plants. Here, we report the crystal structure of the E. coli enzyme at 2.6 A resolution, the first three-dimensional structure of any bacterial Class II KARI. The enzyme consists of two domains, one with mixed alpha/beta structure, which is similar to that found in other pyridine nucleotide-dependent dehydrogenases. The second domain is mainly alpha-helical and shows strong evidence of internal duplication. Comparison of the active sites between KARI of E. coli, Pseudomonas aeruginosa, and spinach shows that most residues occupy conserved positions in the active site. E. coli KARI was crystallized as a tetramer, the likely biologically active unit. This contrasts with P. aeruginosa KARI, which forms a dodecamer, and spinach KARI, a dimer. In the E. coli KARI tetramer, a novel subunit-to-subunit interacting surface is formed by a symmetrical pair of bulbous protrusions.     

Processing enzyme glucosidase II: proposed catalytic residues and developmental regulation during the ontogeny of the mouse mammary gland

Following the action of glucosidase I to clip the terminal alpha1,2-linked glucose, glucosidase II sequentially cleaves the two inner alpha1,3-linked glucose residues from the Glcalpha1,2Glcalpha1,3Glcalpha1,3Man(9)GlcNAc(2) oligosaccharide of the incipient glycoprotein as it undergoes folding and maturation. Glucosidase II belongs to family 31 glycosidases. These enzymes act by the acid-base catalytic mechanism. The cDNA of the wild-type and several mutant forms of the fusion protein of the enzyme in which mutations were introduced in the conserved motif D(564)MNE(567) were expressed in Sf9 cells, and the proteins were purified on Ni-NTA matrix. The catalytic activity of the purified proteins was determined with radioactive Glc(2)Man(9)GlcNAc(2) substrate. The results show that the aspartate and glutamate within the D(564)MNE(567) motif can serve for catalysis, most likely as the acid-base pair within the active site of the enzyme. The developmental regulation of glucosidase II was studied during the ontogeny of the mouse mammary gland for its growth and differentiation. The mRNA of both alpha and beta subunits of the enzyme, immunoreactive alpha and beta subunits, and enzyme activity were measured over the complete developmental cycle. The changes in all the parameters were consistent with similar fluctuations with several other enzymes of the N-glycosylation machinery reported earlier, reaching a three- to fourfold increase over the basal level in the virgin gland at the peak of lactation. Altogether it appears that there is a coordinated regulation of the enzymes involved in protein N-glycosylation during the development of the mouse mammary gland.     

A phosphate-binding subsite in bovine pancreatic ribonuclease A can be converted into a very efficient catalytic site

A general acid-base catalytic mechanism is responsible for the cleavage of the phosphodiester bonds of the RNA by ribonuclease A (RNase A). The main active site is formed by the amino acid residues His12, His119, and Lys41, and the process follows an endonucleolytic pattern that depends on the existence of a noncatalytic phosphate-binding subsite adjacent, on the 3'-side, to the active site; in this region the phosphate group of the substrate establishes electrostatic interactions through the side chains of Lys7 and Arg10. We have obtained, by means of site-directed mutagenesis, RNase A variants with His residues both at positions 7 and 10. These mutations have been introduced with the aim of transforming a noncatalytic binding subsite into a putative new catalytic active site. The RNase activity of these variants was determined by the zymogram technique and steady-state kinetic parameters were obtained by spectrophotometric methods. The variants showed a catalytic efficiency in the same order of magnitude as the wild-type enzyme. However, we have demonstrated in these variants important effects on the substrate's cleavage pattern. The quadruple mutant K7H/R10H/H12K/H119Q shows a clear increase of the exonucleolytic activity; in this case the original native active site has been suppressed, and, as consequence, its activity can only be associated to the new active site. In addition, the mutant K7H/R10H, with two putative active sites, also shows an increase in the exonucleolytic preference with respect to the wild type, a fact that may be correlated with the contribution of the new active site.     

Contribution of active site residues to the activity and thermal stability of ribonuclease Sa

We have used site-specific mutagenesis to study the contribution of Glu 74 and the active site residues Gln 38, Glu 41, Glu 54, Arg 65, and His 85 to the catalytic activity and thermal stability of ribonuclease Sa. The activity of Gln38Ala is lowered by one order of magnitude, which confirms the involvement of this residue in substrate binding. In contrast, Glu41Lys had no effect on the ribonuclease Sa activity. This is surprising, because the hydrogen bond between the guanosine N1 atom and the side chain of Glu 41 is thought to be important for the guanine specificity in related ribonucleases. The activities of Glu54Gln and Arg65Ala are both lowered about 1000-fold, and His85Gln is totally inactive, confirming the importance of these residues to the catalytic function of ribonuclease Sa. In Glu74Lys, k(cat) is reduced sixfold despite the fact that Glu 74 is over 15 A from the active site. The pH dependence of k(cat)/K(M) is very similar for Glu74Lys and wild-type RNase Sa, suggesting that this is not due to a change in the pK values of the groups involved in catalysis. Compared to wild-type RNase Sa, the stabilities of Gln38Ala and Glu74Lys are increased, the stabilities of Glu41Lys, Glu54Gln, and Arg65Ala are decreased and the stability of His85Gln is unchanged. Thus, the active site residues in the ribonuclease Sa make different contributions to the stability.     

乙酰胆碱酯酶分子研究进展

乙酰胆碱酯酶是生物体内胆碱能神经传导中的一种关键酶,它能够快速水解神经递质乙酰胆碱,保证神经信号在生物体内正常传递。乙酰胆碱酯酶结构和催化功能研究一直是生命科学的重要课题,可为研制有针对性的新药和杀虫剂提供重要理论依据。我们简要介绍了乙酰胆碱酯酶的三维晶体结构、活性位点、高效催化机制及其应用。     

Mutational and crystallographic analyses of the active site residues of the Bacillus circulans xylanase.

Using site-directed mutagenesis we have investigated the catalytic residues in a xylanase from Bacillus circulans. Analysis of the mutants E78D and E172D indicated that mutations in these conserved residues do not grossly alter the structure of the enzyme and that these residues participate in the catalytic mechanism. We have now determined the crystal structure of an enzyme-substrate complex to 108 A resolution using a catalytically incompetent mutant (E172C). In addition to the catalytic residues, Glu 78 and Glu 172, we have identified 2 tyrosine residues, Tyr 69 and Tyr 80, which likely function in substrate binding, and an arginine residue, Arg 112, which plays an important role in the active site of this enzyme. On the basis of our work we would propose that Glu 78 is the nucleophile and that Glu 172 is the acid-base catalyst in the reaction.     

Structural clusters of evolutionary trace residues are statistically significant and common in proteins

Given the massive increase in the number of new sequences and structures, a critical problem is how to integrate these raw data into meaningful biological information. One approach, the Evolutionary Trace, or ET, uses phylogenetic information to rank the residues in a protein sequence by evolutionary importance and then maps those ranked at the top onto a representative structure. If these residues form structural clusters, they can identify functional surfaces such as those involved in molecular recognition. Now that a number of examples have shown that ET can identify binding sites and focus mutational studies on their relevant functional determinants, we ask whether the method can be improved so as to be applicable on a large scale. To address this question, we introduce a new treatment of gaps resulting from insertions and deletions, which streamlines the selection of sequences used as input. We also introduce objective statistics to assess the significance of the total number of clusters and of the size of the largest one. As a result of the novel treatment of gaps, ET performance improves measurably. We find evolutionarily privileged clusters that are significant at the 5% level in 45 out of 46 (98%) proteins drawn from a variety of structural classes and biological functions. In 37 of the 38 proteins for which a protein-ligand complex is available, the dominant cluster contacts the ligand. We conclude that spatial clustering of evolutionarily important residues is a general phenomenon, consistent with the cooperative nature of residues that determine structure and function. In practice, these results suggest that ET can be applied on a large scale to identify functional sites in a significant fraction of the structures in the protein databank (PDB). This approach to combining raw sequences and structure to obtain detailed insights into the molecular basis of function should prove valuable in the context of the Structural Genomics Initiative.     

A new mechanism for altering chromosome number during karyotype evolution

Summary A new mechanism for changing chromosome numbers (preserving the fundamental number of long chromosome arms) during karyotype evolution is suggested. It includes: 1) Occurrence of individuals heterozygous for two interchanges between different arms of three chromosomes (a metacentric and two acrocentric ones). 2) Formation in heterokaryotypes of multivalents during meiosis between the chromosomes involved in the interchanges and their unchanged homologues. 3) Mis-segregation of chromosomes from these multivalents resulting in hypoploid (n-1) and hyperploid (n+1) simultaneously instead of euhaploid gametes. 4) Fusion of n-1 or n+1 gametes which gives rise to (zygotes and) individuals representing homokaryotypes with changed number of chromosomes (2n+2 or 2n-2), but preserves (as compared to the parental karyotypes) the number of long chromosome arms. Under definite conditions, chromosome numbers of the progeny may be changed by this process in both directions (upwards and downwards). The mechanism is free of the difficulties associated with the explanation for such changes by direct Robertsonian interchanges (see Discussion), which are usually considered to be responsible for such alterations in chromosome number. The above-mentioned process has been experimentally documented in Vicia faba and it probably also occurred naturally within the Vicia sativa group.     

Ultrahigh resolution structure of a class A beta-lactamase: on the mechanism and specificity of the extended-spectrum SHV-2 enzyme

Bacterial beta-lactamases hydrolyze beta-lactam antibiotics such as penicillins and cephalosporins. The TEM-type class A beta-lactamase SHV-2 is a natural variant that exhibits activity against third-generation cephalosporins normally resistant to hydrolysis by class A enzymes. SHV-2 contains a single Gly238Ser change relative to the wild-type enzyme SHV-1. Crystallographic refinement of a model including hydrogen atoms gave R and R(free) of 12.4% and 15.0% for data to 0.91 A resolution. The hydrogen atom on the O(gamma) atom of the reactive Ser70 is clearly seen for the first time, bridging to the water molecule activated by Glu166. Though hydrogen atoms on the nearby Lys73 are not seen, this observation of the Ser70 hydrogen atom and the hydrogen bonding pattern around Lys73 indicate that Lys73 is protonated. These findings support a role for the Glu166-water couple, rather than Lys73, as the general base in the deprotonation of Ser70 in the acylation process of class A beta-lactamases. Overlay of SHV-2 with SHV-1 shows a significant 1-3 A displacement in the 238-242 beta-strand-turn segment, making the beta-lactam binding site more open to newer cephalosporins with large C7 substituents and thereby expanding the substrate spectrum of the variant enzyme. The OH group of the buried Ser238 side-chain hydrogen bonds to the main-chain CO of Asn170 on the Omega loop, that is unaltered in position relative to SHV-1. This structural role for Ser238 in protein-protein binding makes less likely its hydrogen bonding to oximino cephalosporins such as cefotaxime or ceftazidime.     

生淀粉糖化酶催化位点氨基酸及酶合成调控的初步研究

通过对Rhizopus OR-1UVN菌种所产生淀粉糖化酶在不同底物不同缓冲溶液条件下酶最适pH的测定,推测出该生淀粉糖化酶活力中心催化位点氨基酸是天冬氨酸(Asp)和谷氨酸(Glu)。实验证明5～50mg/mL浓度葡萄糖对生淀粉糖化酶没有抑制作用。分别以浓度<5mg/mL葡萄糖和淀粉为碳源的培养基进行不同碳源发酵实验,发现以淀粉为碳源的培养基Ⅰ发酵15h开始产生淀粉糖化酶,以葡萄糖为碳源的培养基Ⅱ发酵35h开始产酶(葡萄糖浓度<8mg/mL),而且前者菌体较后者少,由此可知葡萄糖对产酶有阻遏作用。实验还发现解阻遏熟淀粉糖化酶的葡萄糖浓度(15mg/mL)比生淀粉糖化酶的要高。由于葡萄糖的阻遏作用不发生在翻译水平,而发生在转录水平上,而且生淀粉糖化酶(G1)与熟淀粉糖化酶(G2)来自同一条DNA链,可以推测存在mRNA的拼接。通过以生淀粉为碳源的比较实验,发现生淀粉对生淀粉糖化酶形成的诱导作用可能主要是通过mRNA拼接的调节来实现的。