Analysis of the active center of branching enzyme II from maize endosperm

Analysis of the primary structure of mBEII, with those of other branching and amylolytic enzymes as reference, identifies four highly conserved regions which may be involved in substrate binding and in catalysis. When one of the amino acid residues corresponding to the putative catalytic sites of mBEII, i.e., Asp-386, Glu-441, and Asp-509, was replaced, activity disappeared. These putative catalytic residues are located in three different regions (regions 2–4) of the four highly conserved regions (regions 1–4) which exist in the primary structure of most starch hydrolases and related enzymes, including branching enzymes. Region 3, which contains Glu-441 as one of the putative catalytic residues, was located downstream of the carboxyl-terminal position previously reported. The importance of the carboxyl amino acid residues was also demonstrated by chemical modification of the branching enzyme protein using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.     

D-serine dehydratase from Escherichia coli. DNA sequence and identification of catalytically inactive glycine to aspartic acid variants

We have identified two glycyl residues whose integrity is essential for the catalytic competence of a model pyridoxal 5'-phosphate requiring enzyme, D-serine dehydratase from Escherichia coli. This was accomplished by isolating and sequencing the structural gene from wild type E. coli and from two mutant strains that produce inactive D-serine dehydratase. DNA sequencing indicated the presence of a single glycine to aspartic acid replacement in each variant. The amino acid replacements lie in a glycine-rich region of D-serine dehydratase well removed from pyridoxal 5'-phosphate-binding lysine 118 in the primary structure of the enzyme. The striking effect of these two glycine to aspartic acid replacements on catalytic activity, the conservation of the glycine-rich region in several pyridoxal 5'-phosphate-dependent enzymes that catalyze alpha/beta-eliminations, and the placement of similar glycine-rich sequences in well-characterized active site structures suggest that the glycine-rich region interacts with the cofactor at the active site of the enzyme.     

Nucleotide sequence of the 3′-terminal region of chilli vein-banding mottle potyvirus isolated from pepper in Thailand

The sequence of the 2227 nucleotides of the 3-terminus of chilli vein-banding mottle virus-Chiangmai isolate (CVbMV-CM1) genome was determined. This sequence encodes a putative 287 amino acid coat protein. Downstream of coat protein coding region is a 288 nucleotide untranslated sequence terminated by a polyadenylate tract. The amino acid sequence of coat protein contained aspartic acid–alanine–glycine tripeptide, which may correlate with the high frequency of aphid transmissibility. The cleavage site between large nuclear inclusion protein (NIB) and coat protein was glutamine/serine. Comparison of 3 non-coding region nucleotide and coat protein amino acid sequences of CVbMV-CM1 with those of other 18 potyviruses showed 44–56% and 54–65% homology respectively, suggesting CVbMV should be regarded as new potyvirus.     

Role for a conserved structural motif in assembly of a class I aminoacyl-tRNA synthetase active site

The catalytic domains of class I aminoacyl-tRNA synthetases are built around a conserved Rossmann nucleotide binding fold, with additional polypeptide domains responsible for tRNA binding or hydrolytic editing of misacylated substrates. Structural comparisons identified a conserved motif bridging the catalytic and anticodon binding domains of class Ia and Ib enzymes. This stem contact fold (SCF) has been proposed to globally orient each enzyme's cognate tRNA by interacting with the inner corner of the L-shaped tRNA. Despite the structural similarity of the SCF among class Ia/Ib enzymes, the sequence conservation is low. We replaced amino acids of the MetRS SCF with portions of the structurally similar glutaminyl-tRNA synthetase (GlnRS) motif or with alanine residues. Chimeric variants retained significant tRNA methionylation activity, indicating that structural integrity of the helix-turn-strand-helix motif contributes more to tRNA aminoacylation than does amino acid identity. In contrast, chimeras were significantly reduced in methionyl adenylate synthesis, suggesting a role for the SCF in formation of a structured active site domain. A highly conserved aspartic acid within the MetRS SCF is proposed to make an electrostatic interaction with an active site lysine; these residues were replaced with alanines or conservative substitutions. Both methionyl adenylate formation and methionine transfer were impaired, and activity was not significantly recovered by making the compensatory double substitution.     

Conserved Interactions of the Active Carboxyls in Pepsin-like Enzymes and Retroviral Proteases

In addition to previous studies, 30 crystal structures of retroviral proteases corresponding to the highest resolution were inspected to analyze the interactions of the active carboxyls with surroundings groups. The outer oxygens of the active carboxyls in retroviral enzymes form contacts only with the water molecule participating in catalysis. This is an important difference between retroviral proteases and pepsin-like enzymes, which form a net of hydrogen bonds of these outer oxygens with residues neighboring the catalytic site in 3D structures. At the same time, it was found that in all aspartic proteases the inner oxygens of the active carboxyls are also involved in the chain of interactions through peptide groups Thr–Gly adjacent to the active residues. Polarization of these peptide groups influences the donor–acceptor properties of the active carboxyls. The found chain of interactions is more extensive in retroviral than in pepsin-like proteases; however, its main part is conserved for the whole class of these enzymes. Some implications of the role of these interactions are discussed.     

Key features determining the specificity of aspartic proteinase inhibition by the helix-forming IA3 polypeptide

The 68-residue IA(3) polypeptide from Saccharomyces cerevisiae is essentially unstructured. It inhibits its target aspartic proteinase through an unprecedented mechanism whereby residues 2-32 of the polypeptide adopt an amphipathic alpha-helical conformation upon contact with the active site of the enzyme. This potent inhibitor (K(i) < 0.1 nm) appears to be specific for a single target proteinase, saccharopepsin. Mutagenesis of IA(3) from S. cerevisiae and its ortholog from Saccharomyces castellii was coupled with quantitation of the interaction for each mutant polypeptide with saccharopepsin and closely related aspartic proteinases from Pichia pastoris and Aspergillus fumigatus. This identified the charged K18/D22 residues on the otherwise hydrophobic face of the amphipathic helix as key selectivity-determining residues within the inhibitor and implicated certain residues within saccharopepsin as being potentially crucial. Mutation of these amino acids established Ala-213 as the dominant specificity-governing feature in the proteinase. The side chain of Ala-213 in conjunction with valine 26 of the inhibitor marshals Tyr-189 of the enzyme precisely into a position in which its side-chain hydroxyl is interconnected via a series of water-mediated contacts to the key K18/D22 residues of the inhibitor. This extensive hydrogen bond network also connects K18/D22 directly to the catalytic Asp-32 and Tyr-75 residues of the enzyme, thus deadlocking the inhibitor in position. In most other aspartic proteinases, the amino acid at position 213 is a larger hydrophobic residue that prohibits this precise juxtaposition of residues and eliminates these enzymes as targets of IA(3). The exquisite specificity exhibited by this inhibitor in its interaction with its cognate folding partner proteinase can thus be readily explained.     

Enhancement of hydrolytic activity of thermophilic alkalophilic α-amylase from Bacillus sp. AAH-31 through optimization of amino acid residues surrounding the substrate binding site

The hydrolytic activity of a thermophilic alkalophilic α-amylase from Bacillus sp. AAH-31 (AmyL) toward soluble starch was enhanced through optimization of amino acid (aa) residues situated near the substrate binding site. Twenty-four selected aa residues were replaced with Ala, and Gly429 and Gly550 were altered to Lys and Glu, respectively, based on comparison of AmyL's aa sequence with related enzymes. Y426A, H431A, I509A, and K549A showed notably higher activity than the wild type at 162–254% of wild-type activity. Tyr426, His431, and Ile509 were predicted to be located near subsite −2, while Lys549 was near subsite +2. Ser, Ala, Ala, and Met were found to be the best aa residues for the positions of Tyr426, His431, Ile509, and Lys549, respectively. Combinations of the optimized single mutations at distant positions were effective in enhancing catalytic activity. The double-mutant enzymes Y426S/K549M, H431A/K549M, and I509A/K549M, combining two of the selected single mutations, showed 340%, 252%, and 271% of wild type activity, respectively. Triple and quadruple-mutant enzymes of the selected mutations did not show higher activity than the best double-mutant, Y426S/K549M.     

The alpha subunit of tryptophan synthase. Evidence that aspartic acid 60 is a catalytic residue and that the double alteration of residues 175 and 211 in a second-site revertant restores the proper geometry of the substrate binding site

Our studies, which are aimed at understanding the catalytic mechanism of the alpha subunit of tryptophan synthase from Salmonella typhimurium, use site-directed mutagenesis to explore the functional roles of aspartic acid 60, tyrosine 175, and glycine 211. These residues are located close to the substrate binding site of the alpha subunit in the three-dimensional structure of the tryptophan synthase alpha 2 beta 2 complex. Our finding that replacement of aspartic acid 60 by asparagine, alanine, or tyrosine results in complete loss of activity in the reaction catalyzed by the alpha subunit supports a catalytic role for aspartic acid 60. Since the mutant form with glutamic acid at position 60 has partial activity, glutamic acid 60 may serve as an alternative catalytic base. The mutant form in which tyrosine 175 is replaced by phenylalanine has substantial activity; thus the phenolic hydroxyl of tyrosine 175 is not essential for catalysis or substrate binding. Yanofsky and colleagues have identified many missense mutant forms of the alpha subunit of tryptophan synthase from Escherichia coli. Two of these inactive mutant forms had either tyrosine 175 replaced by cysteine or glycine 211 replaced by glutamic acid. Surprisingly, a second-site revertant which contained both of these amino acid changes was partially active. These results indicated that the second mutation must compensate in some way for the first. We now extend the studies of the effects of specific amino acid replacements at positions 175 and 211 by two techniques: 1) characterization of several mutant forms of the alpha subunit from S. typhimurium prepared by site-directed mutagenesis and 2) computer graphics modeling of the substrate binding site of the alpha subunit using the x-ray coordinates of the wild type alpha 2 beta 2 complex from S. typhimurium. We conclude that the restoration of alpha subunit activity in the doubly altered second-site revertant results from restoration of the proper geometry of the substrate binding site.     

Crystal structure of cockroach allergen Bla g 2, an unusual zinc binding aspartic protease with a novel mode of self-inhibition

The crystal structure of Bla g 2 was solved in order to investigate the structural basis for the allergenic properties of this unusual protein. This is the first structure of an aspartic protease in which conserved glycine residues, in two canonical DTG triads, are substituted by different amino acid residues. Another unprecedented feature revealed by the structure is the single phenylalanine residue insertion on the tip of the flap, with the side-chain occupying the S1 binding pocket. This and other important amino acid substitutions in the active site region of Bla g 2 modify the interactions in the vicinity of the catalytic aspartate residues, increasing the distance between them to approximately 4A and establishing unique direct contacts between the flap and the catalytic residues. We attribute the absence of substantial catalytic activity in Bla g 2 to these unusual features of the active site. Five disulfide bridges and a Zn-binding site confer stability to the protein, which may contribute to sensitization at lower levels of exposure than other allergens.     

The roles of highly conserved,non‐catalytic residues in class A β‐lactamases

Evolution minimizes the number of highly conserved amino acid residues in proteins to ensure evolutionary robustness and adaptability. The roles of all highly conserved, non‐catalytic residues, 11% of all residues, in class A β‐lactamase were analyzed by studying the effect of 146 mutations on in cell and in vitro activity, folding, structure, and stability. Residues around the catalytic residues (second shell) contribute to fine‐tuning of the active site structure. Mutations affect the structure over the entire active site and can result in stable but inactive protein. Conserved residues farther away (third shell) ensure a favorable balance of folding versus aggregation or stabilize the folded form over the unfolded state. Once folded, the mutant enzymes are stable and active and show only localized structural effects. These residues are found in clusters, stapling secondary structure elements. The results give an integral picture of the different roles of essential residues in enzymes.     

Characterization of an active single polypeptide form of the human immunodeficiency virus type 1 protease

The pepsin-like aspartyl proteases consist of a single polypeptide chain with topologically similar amino- and carboxyl-terminal domains, each of which contributes 1 aspartic acid residue to the active site. This structure has been proposed to have evolved by gene duplication and fusion from a dimeric enzyme composed of two identical polypeptide chains, such as the aspartyl protease (PRT) of human immunodeficiency virus type 1 (HIV-1). To determine if a single polypeptide form of the HIV-1 protease would be enzymatically active, two protease coding regions were linked to form a dimeric gene (pFGGP). Expression of this gene in Escherichia coli yielded a protein with the expected molecular mass of 22 kDa. The in vitro kinetic parameters of PRT and FGGP (where FGGP is the single polypeptide form of the HIV-1 protease with 2 glycine residues connecting the two subunits) for three peptide substrates are similar. Construction and analysis of a CheY-GAG-FGGP fusion protein demonstrated that FGGP is capable of precursor processing in vivo. Mutation of one or both of the active site aspartates to either asparagine or glutamate rendered the enzyme inactive, demonstrating that both active site aspartate residues are required for enzymatic activity.     

Relationship of sequence and structure to specificity in the alpha-amylase family of enzymes

The hydrolases and transferases that constitute the alpha-amylase family are multidomain proteins, but each has a catalytic domain in the form of a (beta/alpha)(8)-barrel, with the active site being at the C-terminal end of the barrel beta-strands. Although the enzymes are believed to share the same catalytic acids and a common mechanism of action, they have been assigned to three separate families - 13, 70 and 77 - in the classification scheme for glycoside hydrolases and transferases that is based on amino acid sequence similarities. Each enzyme has one glutamic acid and two aspartic acid residues necessary for activity, while most enzymes of the family also contain two histidine residues critical for transition state stabilisation. These five residues occur in four short sequences conserved throughout the family, and within such sequences some key amino acid residues are related to enzyme specificity. A table is given showing motifs distinctive for each specificity as extracted from 316 sequences, which should aid in identifying the enzyme from primary structure information. Where appropriate, existing problems with identification of some enzymes of the family are pointed out. For enzymes of known three-dimensional structure, action is discussed in terms of molecular architecture. The sequence-specificity and structure-specificity relationships described may provide useful pointers for rational protein engineering.     

Tryptophanase in aqueous methanol: the solvent effects and a probable mechanism of the hydrophobic control of substrate specificity

To shed light on the mechanism of hydrophobic control in reactions of microbial tryptophanase the direct effect of the solvent hydrophobicity on affinities of amino acid inhibitors was first examined. Values of inhibition constants (K_i) for a variety of amino acids were determined in 37.5% aqueous methanol, and no general correlation between the change of K_i, on passing from water to aqueous methanol, and amino acid hydrophobicity was found. The solvent effects on the separate stages of the external aldimine formation (K_D) and deprotonation to form a quinonoid intermediate (K_q) were determined for the reactions of tryptophanase with 2-oxindolyl- -alanine and -alanine by stopped-flow technique. For 2-oxindolyl- -alanine, which is a close transition-state analogue for the enzyme reaction with natural substrate, the decrease in the affinity in aqueous methanol is associated exclusively with the α-proton abstraction stage but not with the preceding formation of external aldimine. We conclude that the environment of amino acid side chains in the active site cannot be considered to be permanently hydrophobic irrespective of the bound amino acid. We suggest that complexes of tryptophanase with amino acids may exist either in a hydrophobic, presumably “closed”, conformation, where bound amino acids are isolated from the solvent, or in an accesible to solvent, “open”, conformation, depending on the structure of the bound amino acid and stage of the catalytic mechanism. For 2-oxindolyl- -alanine the transfer from an open to a closed conformation probably accompanies deprotonation of the external aldimine. The change of the active site hydrophobicity may provide an efficient way of modulating the relative acid–base properties of the catalytic groups to ensure the movement of protons in the “correct” direction depending on the elementary stage of catalysis.     

Possible involvement of glutamic and/or aspartic acid residue(s) and requirement of mitochondrial integrity for the protective effect of creatine against inhibition of cardiac mitochondrial respiration by methylglyoxal

We had previously shown that creatine exerted a protective effect against inhibition of cardiac mitochondrial respiration by methylglyoxal (SinhaRoy S, Biswas S, Ray M, Ray S. Biochem J 372: 661–669, 2003). In the present study, we have investigated the mechanism of this protective effect by specific amino acid modifying reagent and by several compounds, which are structurally related to creatine. The results show that the compounds, which contain guanidine group such as arginine and guanidinopropionic acid, exert a protective effect, which is quantitatively similar to creatine. This result suggests the presence of carboxylic acid(s) such as glutamic and/or aspartic acid(s) in the creatine-binding site, which has been further supported by experiments with N-ethyl-5-phenyl isoxazolium-3-sulfonate a reagent known to modify these amino acids. Both polarographic and spectrophotometric assays were performed with NADH as respiratory substrate by using a) submitochondrial particles by sonication, b) freeze-thawed mitochondria and c) mitochondria permeabilized by alamethicin treatment. The results of these studies as compared to that of intact mitochondria indicate that structural integrity of mitochondria is essential for the protective effect of creatine. (Mol Cell Biochem 271: 167–176, 2005)     

Binding residues and catalytic domain of soluble Saccharomyces cerevisiae processing alpha-glucosidase I

Alpha-glucosidase I initiates the trimming of newly assembled N-linked glycoproteins in the lumen of the endoplasmic reticulum (ER). Site-specific chemical modification of the soluble alpha-glucosidase I from yeast using diethylpyrocarbonate (DEPC) and tetranitromethane (TNM) revealed that histidine and tyrosine are involved in the catalytic activity of the enzyme, as these residues could be protected from modification using the inhibitor deoxynojirimycin. Deoxynojirimycin could not prevent inactivation of enzyme treated with N-bromosuccinimide (NBS) used to modify tryptophan residues. Therefore, the binding mechanism of yeast enzyme contains different amino acid residues compared to its mammalian counterpart. Catalytically active polypeptides were isolated from endogenous proteolysis and controlled trypsin hydrolysis of the enzyme. A 37-kDa nonglycosylated polypeptide was isolated as the smallest active fragment from both digests, using affinity chromatography with inhibitor-based resins (N-methyl-N-59-carboxypentyl- and N-59-carboxypentyl-deoxynojirimycin). N-terminal sequencing confirmed that the catalytic domain of the enzyme is located at the C-terminus. The hydrolysis sites were between Arg(521) and Thr(522) for endogenous proteolysis and residues Lys(524) and Phe(525) for the trypsin-generated peptide. This 37-kDa polypeptide is 1.9 times more active than the 98-kDa protein when assayed with the synthetic trisaccharide, alpha-D-Glc1,2alpha-D-Glc1,3alpha-D-Glc-O(CH2)(8)COOCH(3), and is not glycosylated. Identification of this relatively small fragment with catalytic activity will allow mechanistic studies to focus on this critical region and raises interesting questions about the relationship between the catalytic region and the remaining polypeptide.     

Structure of the Homodimeric Glycine Decarboxylase P-protein from Synechocystis sp. PCC 6803 Suggests a Mechanism for Redox Regulation

Glycine decarboxylase, or P-protein, is a pyridoxal 5′-phosphate (PLP)-dependent enzyme in one-carbon metabolism of all organisms, in the glycine and serine catabolism of vertebrates, and in the photorespiratory pathway of oxygenic phototrophs. P-protein from the cyanobacterium Synechocystis sp. PCC 6803 is an α₂ homodimer with high homology to eukaryotic P-proteins. The crystal structure of the apoenzyme shows the C terminus locked in a closed conformation by a disulfide bond between Cys⁹⁷² in the C terminus and Cys³⁵³ located in the active site. The presence of the disulfide bridge isolates the active site from solvent and hinders the binding of PLP and glycine in the active site. Variants produced by substitution of Cys⁹⁷² and Cys³⁵³ by Ser using site-directed mutagenesis have distinctly lower specific activities, supporting the crucial role of these highly conserved redox-sensitive amino acid residues for P-protein activity. Reduction of the 353–972 disulfide releases the C terminus and allows access to the active site. PLP and the substrate glycine bind in the active site of this reduced enzyme and appear to cause further conformational changes involving a flexible surface loop. The observation of the disulfide bond that acts to stabilize the closed form suggests a molecular mechanism for the redox-dependent activation of glycine decarboxylase observed earlier.     

The Proteolytic Activity and Cleavage Specificity of Fibronectin-Gelatinase and Fibronectin-Lamininase

Human plasma fibronectin contains two latent aspartic proteinases, FN-gelatinase and FN-lamininase. Both enzymes can be generated and activated in the presence of Ca²⁺ from the purified cathepsin D-produced 190-kDa fibronectin fragment. We investigated the proteolytic activity and cleavage specificity of both enzymes in a range of pH from 3.5 to 9.0 using the B chain of oxidized bovine insulin and chromogenic peptides as substrates. The inhibition of the enzymes by several natural inhibitors from human plasma was also tested. The specificities of FN-gelatinase and FN-lamininase are similar to other major acidic proteinases, including pepsin, renin, cathepsin D, and HIV-proteinases. Both enzymes mainly hydrolyze three peptide bonds in the oxidized insulin B chain, namely Glu–Ala (residues 13–14), Tyr–Leu (residues 16–17), and Phe–Phe (residues 24–25). For the peptide substrates H-Pro-Thr-Glu-Phe-p-nitro-Phe-Arg-Leu-OH and H-Phe-Gly-His-p-nitro-Phe-Phe-Val-Leu-OMe that were cleaved the respective values of k _cat/K _M were 105.1 and 11.8 mM^–1 sec^–1 for cleavage by FN-gelatinase, and 123.2 and 15.5 mM^–1 sec^–1 for cleavage by FN-lamininase. The maximal activities of both enzymes were observed in a range between pH 5.6 and 6.3 and they became inactivated at a pH value above 8.4. Both FN-gelatinase and FN-lamininase were efficiently inhibited by ₂-macroglobulin.     

Structural Basis for Evolution of Product Diversity in Soybean Glutathione Biosynthesis

The redox active peptide glutathione is ubiquitous in nature, but some plants also synthesize glutathione analogs in response to environmental stresses. To understand the evolution of chemical diversity in the closely related enzymes homoglutathione synthetase (hGS) and glutathione synthetase (GS), we determined the structures of soybean (Glycine max) hGS in three states: apoenzyme, bound to γ-glutamylcysteine (γEC), and with hGSH, ADP, and a sulfate ion bound in the active site. Domain movements and rearrangement of active site loops change the structure from an open active site form (apoenzyme and γEC complex) to a closed active site form (hGSH•ADP•SO₄²⁻ complex). The structure of hGS shows that two amino acid differences in an active site loop provide extra space to accommodate the longer β-Ala moiety of hGSH in comparison to the glycinyl group of glutathione. Mutation of either Leu-487 or Pro-488 to an Ala improves catalytic efficiency using Gly, but a double mutation (L487A/P488A) is required to convert the substrate preference of hGS from β-Ala to Gly. These structures, combined with site-directed mutagenesis, reveal the molecular changes that define the substrate preference of hGS, explain the product diversity within evolutionarily related GS-like enzymes, and reinforce the critical role of active site loops in the adaptation and diversification of enzyme function.     

Copper reduction by copper binding proteins and its relation to neurodegenerative diseases

Increasing evidence supports an important role for metals in neurobiology. In fact, copper binding proteins that form bioinorganic complexes are able to display oxidant or anti-oxidant properties, which would impact on neuronal function or in the triggering of neurodegenerative process. Two proteins related to neurodegenerative diseases have been described as copper binding proteins: the amyloid precursor protein (APP), a protein related to Alzheimer's disease, and the Prion protein (PrP), related to Creutzfeldt-Jakob disease. We used different synthetic peptides from APP and PrP sequences in order to evaluate the ability to reduce copper. We observed that APP_135–156, amyloid--peptide (A_1–40), and PrP_59–91 all have copper reducing ability, with the APP_135–156 peptide being more potent than the other fragments. Moreover, we identify His, Cys and Trp residues as key amino acids involved in the copper reduction of A, APP and PrP, respectively. We postulated, that in a cellular context, the interaction of these proteins with copper could be necessary to reduce copper on plasma membrane, possibly presenting Cu(I) to the copper transporter, driving the delivery of this metal to antioxidant enzymes. Moreover, protein-metal complexes could be the catalytic centers for the formation of reactive oxygen species involved in the oxidative damage present both in Alzheimer's and Prion disease.