Fragmentation of cellulose by the major Thermomonospora fusca cellulases, Trichoderma reesei CBHI, and their mixtures

In this study, the fragmentation activities of Thermomonospora fusca cellulases E(2), E(3), E(5), Trichoderma reesei CBHI, and their mixtures were measured to study synergism in fragmentation. Fragmentation studies revealed that only two pure cellulases, T. fusca E(2) and E(5) had significant fragmentation activity. T. fusca E(3) shows strong synergism in fragmentation both in the production of reducing sugars and in fragmentation with both T. fusca endoglucananses and with T. reesei CBHI. Most mixtures containing CBHI produced higher rates of fragmentation than comparable mixtures containing E(3). The highest rate and extent of reducing sugar formation and the highest fragmentation activity were obtained with a mixture of E(2), E(3), and CBHI. (c) 1992 John Wiley & Sons, Inc.     

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.     

Possible structure and active site residues of starch,glycogen, and sucrose synthases

A group of enzymes that include muscle glycogen phosphorylase and sugar transferases involved in, for example, the glucosylation of DNA and the synthesis of peptidoglycan are known to possess the same basic three-dimensional fold. Here the possibility is examined that other monosaccharide transferases, those that catalyze synthesis of starch, glycogen, and the disaccharide sucrose, resemble the phosphorylase-type enzymes in structure. In particular, a clear relationship is shown, for the first time, between mammalian glycogen synthases and the phosphorylase structural group of proteins. Domain architecture and secondary structure are discussed, and the possible role of several conserved amino acids at the active site is explored.     

Cel9D, an atypical 1,4-beta-D-glucan glucohydrolase from Fibrobacter succinogenes: characteristics, catalytic residues, and synergistic interactions with other cellulases

The increasing demands of renewable energy have led to the critical emphasis on novel enzymes to enhance cellulose biodegradation for biomass conversion. To identify new cellulases in the ruminal bacterium Fibrobacter succinogenes, a cell extract of cellulose-grown cells was separated by ion-exchange chromatography and cellulases were located by zymogram analysis and identified by peptide mass fingerprinting. An atypical family 9 glycoside hydrolase (GH9), Cel9D, with less than 20% identity to typical GH9 cellulases, was identified. Purified recombinant Cel9D enhanced the production of reducing sugar from acid swollen cellulose (ASC) and Avicel by 1.5- to 4-fold when mixed separately with each of four other glucanases, although it had low activity on these substrates. Cel9D degraded ASC and cellodextrins with a degree of polymerization higher than 2 to glucose with no apparent endoglucanase activity, and its activity was restricted to beta-1-->4-linked glucose residues. It catalyzed the hydrolysis of cellulose by an inverting mode of reaction, releasing glucose from the nonreducing end. Unlike many GH9 cellulases, calcium ions were not required for its function. Cel9D had increased kcat/Km values for cello-oligosaccharides with higher degrees of polymerization. The kcat/Km value for cellohexaose was 2,300 times higher than that on cellobiose. This result indicates that Cel9D is a 1,4-beta-D-glucan glucohydrolase (EC 3.2.1.74) in the GH9 family. Site-directed mutagenesis of Cel9D identified Asp166 and Glu612 as the candidate catalytic residues, while Ser168, which is not present in typical GH9 cellulases, has a crucial structural role. This enzyme has an important role in crystalline cellulose digestion by releasing glucose from accessible cello-oligosaccharides.     

An active site rearrangement within the Tetrahymena group I ribozyme releases nonproductive interactions and allows formation of catalytic interactions

Biological catalysis hinges on the precise structural integrity of an active site that binds and transforms its substrates and meeting this requirement presents a unique challenge for RNA enzymes. Functional RNAs, including ribozymes, fold into their active conformations within rugged energy landscapes that often contain misfolded conformers. Here we uncover and characterize one such “off-pathway” species within an active site after overall folding of the ribozyme is complete. The Tetrahymena group I ribozyme (E) catalyzes cleavage of an oligonucleotide substrate (S) by an exogenous guanosine (G) cofactor. We tested whether specific catalytic interactions with G are present in the preceding E•S•G and E•G ground-state complexes. We monitored interactions with G via the effects of 2′- and 3′-deoxy (–H) and −amino (–NH₂) substitutions on G binding. These and prior results reveal that G is bound in an inactive configuration within E•G, with the nucleophilic 3′-OH making a nonproductive interaction with an active site metal ion termed M_A and with the adjacent 2′-OH making no interaction. Upon S binding, a rearrangement occurs that allows both –OH groups to contact a different active site metal ion, termed M_C, to make what are likely to be their catalytic interactions. The reactive phosphoryl group on S promotes this change, presumably by repositioning the metal ions with respect to G. This conformational transition demonstrates local rearrangements within an otherwise folded RNA, underscoring RNA''s difficulty in specifying a unique conformation and highlighting Nature''s potential to use local transitions of RNA in complex function.     

Mutations of fumarase that distinguish between the active site and a nearby dicarboxylic acid binding site.

Two mutant forms of fumarase C from E. coli have been made using PCR and recombinant DNA. The recombinant form of the protein included a histidine arm on the C-terminal facilitating purification. Based on earlier studies, two different carboxylic acid binding sites, labeled A- and B-, were observed in crystal structures of the wild type and inhibited forms of the enzyme. A histidine at each of the sites was mutated to an asparagine. H188N at the A-site resulted in a large decrease in specific activity, while the H129N mutation at the B-site had essentially no effect. From the results, we conclude that the A-site is indeed the active site, and a dual role for H188 as a potential catalytic base is proposed. Crystal structures of the two mutant proteins produced some unexpected results. Both mutations reduced the affinity for the carboxylic acids at their respective sites. The H129N mutant should be particularly useful in future kinetic studies because it sterically blocks the B-site with the carboxyamide of asparagine assuming the position of the ligand's carboxylate. In the H188N mutation at the active site, the new asparagine side chain still interacts with an active site water that appears to have moved slightly as a result of the mutation.     

Synergistic activity of Paenibacillus sp. BP-23 cellobiohydrolase Cel48C in association with the contiguous endoglucanase Cel9B and with endo- or exo-acting glucanases from Thermobifida fusca

Cellobiohydrolase Cel48C from Paenibacillus sp. BP-23, an enzyme displaying limited activity on most cellulosic substrates, was assayed for activity in the presence of other bacterial endo- or exocellulases. Significant enhanced activity was observed when Cel48C was incubated in the presence of Paenibacillus sp. BP-23 endoglucanase Cel9B or Thermobifida fusca cellulases Cel6A and Cel6B, indicating that Cel48C acts synergistically with them. Maximum synergism rates on bacterial microcrystalline cellulose or filter paper were obtained with a mixture of Paenibacillus cellulases Cel9B and Cel48C, accompanied by T. fusca exocellulase Cel6B. Synergism was also observed in cell extracts from recombinant clone E. coli pUCel9-Cel48 expressing the two contiguous Paenibacillus cellulases Cel9B and Cel48C. The enhanced cellulolytic activity displayed by the cellulase mixtures assayed could be used as an efficient tool for biotechnological applications like pulp and paper manufacturing.     

A rapid and direct method for the determination of active site accessibility in proteins based on ESI-MS and active site titrations

We have developed an electrospray ionisation mass spectrometry (ESI-MS) technique that can be applied to rapidly determine the number of intact active sites in proteins. The methodology relies on inhibiting the protein with an active-site irreversible inhibitor and then using ESI-MS to determine the extent of inhibition. We have applied this methodology to a test system: a serine protease, subtilisin Carlsberg, and monitored the extent of inhibition by phenylmethylsulfonyl fluoride (PMSF), an irreversible serine hydrolase inhibitor as a function of the changes in immobilisation and hydration conditions. Two types of enzyme preparation were investigated, lyophilised enzymes and protein-coated microcrystals (PCMC).     

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。     

X-ray crystallographic analysis of the structural basis for the interactions of pokeweed antiviral protein with its active site inhibitor and ribosomal RNA substrate analogs.

The pokeweed antiviral protein (PAP) belongs to a family of ribosome-inactivating proteins (RIP), which depurinate ribosomal RNA through their site-specific N-glycosidase activity. We report low temperature, three-dimensional structures of PAP co-crystallized with adenyl-guanosine (ApG) and adenyl-cytosine-cytosine (ApCpC). Crystal structures of 2.0-2.1 A resolution revealed that both ApG or ApCpC nucleotides are cleaved by PAP, leaving only the adenine base clearly visible in the active site pocket of PAP. ApCpC does not resemble any known natural substrate for any ribosome-inactivating proteins and its cleavage by PAP provides unprecedented evidence for a broad spectrum N-glycosidase activity of PAP toward adenine-containing single stranded RNA. We also report the analysis of a 2.1 A crystal structure of PAP complexed with the RIP inhibitor pteoric acid. The pterin ring is strongly bound in the active site, forming four hydrogen bonds with active site residues and one hydrogen bond with the coordinated water molecule. The second 180 degrees rotation conformation of pterin ring can form only three hydrogen bonds in the active site and is less energetically favorable. The benzoate moiety is parallel to the protein surface of PAP and forms only one hydrogen bond with the guanido group of Arg135.     

Effects of active site cleft residues on oligosaccharide binding,hydrolysis, and glycosynthase activities of rice BGlu1 and its mutants

Rice BGlu1 (Os3BGlu7) is a glycoside hydrolase family 1 β‐glucosidase that hydrolyzes cellooligosaccharides with increasing efficiency as the degree of polymerization (DP) increases from 2 to 6, indicating six subsites for glucosyl residue binding. Five subsites have been identified in X‐ray crystal structures of cellooligosaccharide complexes with its E176Q acid‐base and E386G nucleophile mutants. X‐ray crystal structures indicate that cellotetraose binds in a similar mode in BGlu1 E176Q and E386G, but in a different mode in the BGlu1 E386G/Y341A variant, in which glucosyl residue 4 (Glc4) interacts with Q187 instead of the eliminated phenolic group of Y341. Here, we found that the Q187A mutation has little effect on BGlu1 cellooligosaccharide hydrolysis activity or oligosaccharide binding in BGlu1 E176Q, and only slight effects on BGlu1 E386G glycosynthase activity. X‐ray crystal structures showed that cellotetraose binds in a different position in BGlu1 E176Q/Y341A, in which it interacts directly with R178 and W337, and the Q187A mutation had little effect on cellotetraose binding. Mutations of R178 and W337 to A had significant and nonadditive effects on oligosaccharide hydrolysis by BGlu1, pNPGlc cleavage and cellooligosaccharide inhibition of BGlu1 E176Q and BGlu1 E386G glycosynthase activity. Hydrolysis activity was partially rescued by Y341 for longer substrates, suggesting stacking of Glc4 on Y341 stabilizes binding of cellooligosaccharides in the optimal position for hydrolysis. This analysis indicates that complex interactions between active site cleft residues modulate substrate binding and hydrolysis.     

Conserved residues within the putative active site of gamma-secretase differentially influence enzyme activity and inhibitor binding

Gamma-secretase performs the final processing step in the generation of amyloid-beta (Abeta) peptides, which are believed to be causative for Alzheimer's disease. Presenilins (PS) are required for gamma-secretase activity and the presence of two essential intramembranous aspartates (D257 and D385) has implicated this region as the putative catalytic centre of an aspartyl protease. The presence of several key hydrogen-bonding residues around the active site of classical aspartyl proteases led us to investigate the role of both the critical aspartates and two nearby conserved hydrogen bond donors in PS1. Generation of cell lines stably overexpressing the D257E, D385E, Y256F and Y389F engineered mutations has enabled us to determine their role in enzyme catalysis and binding of a transition state analogue gamma-secretase inhibitor. Here we report that replacement of either tyrosine residue alters gamma-secretase cleavage specificity, resulting in an increase in the production of the more pathogenic Abeta42 peptide in both cells and membranous enzyme preparations, without affecting inhibitor binding. In contrast, replacement of either of the aspartate residues precludes inhibitor binding in addition to inactivation of the enzyme. Together, these data further incriminate the region around the intramembranous aspartates as the active site of the enzyme, targeted by transition state analogue inhibitors, and highlight the roles of individual residues.     

Ionogenic groups in the active site of lysostaphin. Kinetic and thermodynamic data compared with X-ray crystallographic data

The active site of lysostaphin is shown to contain a residue of glutamic acid. As judged by a pK value of 9.2 (with pentaglycine bridges in peptidoglycan of staphylococci as a substrate), another ionogenic residue could be the epsilon-amino group of a lysine. However, the pH value near a negatively charged cell is supposed to be strongly shifted to acidity as compared to the pH of the solution volume. This shifts the enzyme pH dependence curve in solution to alkalinity. Therefore, the other group might be histidine, which is consistent with the X-ray crystallographic data. A similar shift is likely to occur for lysozyme in the case of Micrococcus lysodeikticus cells. Determination of pK of ionogenic groups in the active sites of alkaline enzymes responsible for lysis of negatively charged bacterial cells gives their apparent values because the "pericellular" and "voluminous" values of pH are not coincident.     

Rapid binding of a cationic active site inhibitor to wild type and mutant mouse acetylcholinesterase: Brownian dynamics simulation including diffusion in the active site gorge

It is known that anionic surface residues play a role in the long-range electrostatic attraction between acetylcholinesterase and cationic ligands. In our current investigation, we show that anionic residues also play an important role in the behavior of the ligand within the active site gorge of acetylcholinesterase. Negatively charged residues near the gorge opening not only attract positively charged ligands from solution to the enzyme, but can also restrict the motion of the ligand once it is inside of the gorge. We use Brownian dynamics techniques to calculate the rate constant k_on for wild type and mutant acetylcholinesterase with a positively charged ligand. These calculations are performed by allowing the ligand to diffuse within the active site gorge. This is an extension of previously reported work in which a ligand was allowed to diffuse only to the enzyme surface. By setting the reaction criteria for the ligand closer to the active site, better agreement with experimental data is obtained. Although a number of residues influence the movement of the ligand within the gorge, Asp74 is shown to play a particularly important role in this function. Asp74 traps the ligand within the gorge, and in this way helps to ensure a reaction. © 1998 John Wiley & Sons, Inc. Biopoly 46: 465–474, 1998     

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.     

Mechanistic studies on carboxypeptidase A from goat pancreas Part I: Role of tyrosine residue at the active site

Chemical modification of carboxypeptidase Ag₁ from goat pancreas with Nacetylimidazole or iodine led to loss of enzymic activity. This loss in activity could be prevented when chemical modification was carried out in the presence of Β-phenylpropionic acid or substrate NCbz-glycyl-L-phenylalanine, thus suggesting a tyrosine residue at the active site. Chemical modification of tyrosine was confirmed by spectral and kinetic studies. While tyrosine modification destroyed peptidase activity, esterase activity of the enzyme remained unchanged thus indicating non-involvement of tyrosine residue in ester hydrolysis     

Exploring the drug resistance mechanism of active site,non-active site mutations and their cooperative effects in CRF01_AE HIV-1 protease: molecular dynamics simulations and free energy calculations

Human immunodeficiency virus type 1 protease is essential for virus replication and maturation and has been considered as one of the important drug target for the antiretroviral treatment of HIV infection. The majority of HIV infections are caused due to non-B subtypes in developing countries. Subtype AE is spreading rapidly and infecting huge population worldwide. Understanding the interdependence of active and non-active site mutations in conferring drug resistance is crucial for the development effective inhibitors in subtype AE protease. In this work, we have investigated the mechanism of resistance against indinavir (IDV) due to therapy selected active site mutation V82F, non-active site mutations PF82V and their cooperative effects PV82F in subtype AE-protease using molecular dynamics simulations and binding free energy calculations. The simulations suggested all the three complexes lead to decrease in binding affinity of IDV, whereas the PF82V complex resulted in an enhanced binding affinity compared to V82F and PV82F complexes. Large positional deviation of IDV was observed in V82F complex. The preservation of hydrogen bonds of IDV with active site Asp25/Asp25′ and flap residue Ile50/50′ via a water molecule is crucial for effective binding. Owing to the close contact of 80s loop with Ile50′ and Asp25, the alteration between residues Thr80 and Val82, further induces conformational change thereby resulting in loss of interactions between IDV and the residues in the active site cavity, leading to drug resistance. Our present study shed light on the effect of active, non-active site mutations and their cooperative effects in AE protease.

Communicated by Ramaswamy H. Sarma     

Modification of the heme active site to increase the peroxidase activity of thermophilic cytochrome P450: A rational approach

The site specific mutants of the thermophilic P450 (P450 175A1 or CYP175A1) were designed to introduce residues that could act as acid-base catalysts near the active site to enhance the peroxidases activity. The Leu80 in the distal heme pocket of CYP175A1 was located at a position almost equivalent to the Glu183 that is involved in stabilization of the ferryl heme intermediate in chloroperoxidase (CPO). The Leu80 residue of CYP175A1 was mutated with histidine (L80H) and glutamine (L80Q) that could potentially form hydrogen bond with hydrogen peroxide and facilitate formation and stabilization of the putative redox intermediate of the peroxidase cycle. The mutants L80H and L80Q of CYP175A1 showed higher peroxidase activity compared to that of the wild type (WT) CYP175A1 enzyme at 25 °C. The activity constants (k_cat) for the L80H and L80Q mutants of CYP175A1 were higher than those of myoglobin and wild type cytochrome b562 at 25 °C. The optimum temperature for the peroxidase activity of the WT and mutants of CYP175A1 was ~ 70 °C. The rate of catalysis at temperatures above ~ 70 °C was higher for L80Q mutant of CYP175A1 compared to that of the well known natural peroxidase, horseradish peroxidase (HRP) that denatures at such high temperature. The peroxidase activities of the mutants of CYP175A1 were maximum at pH 9, unlike that of HRP which is at pH ~ 5. The results have been discussed in the light of understanding the structure-function relationship of the peroxidase properties of these thermostable heme proteins.     

Identification of the catalytic subdomain of the VS ribozyme and evidence for remarkable sequence tolerance in the active site loop

We show here that the ribozyme domain of the Neurospora VS ribozyme consists of separable upper and lower subdomains. Deletion analysis demonstrates that the entire upper subdomain (helices III/IV/V) is dispensable for site-specific cleavage activity, providing experimental evidence that the active site is contained within the lower subdomain and within the substrate itself. We demonstrate an important role in cleavage activity for a region of helix VI called the 730 loop. Surprisingly, several loop sequences, sizes, and structures at this position can support site-specific cleavage, suggesting that a variety of non-Watson-Crick structures, rather than a specific loop structure, in this region of the ribozyme can contribute to formation of the active site.