The role in the substrate specificity and catalysis of residues forming the substrate aglycone-binding site of a beta-glycosidase

The relative contributions to the specificity and catalysis of aglycone, of residues E190, E194, K201 and M453 that form the aglycone-binding site of a beta-glycosidase from Spodoptera frugiperda (EC 3.2.1.21), were investigated through site-directed mutagenesis and enzyme kinetic experiments. The results showed that E190 favors the binding of the initial portion of alkyl-type aglycones (up to the sixth methylene group) and also the first glucose unit of oligosaccharidic aglycones, whereas a balance between interactions with E194 and K201 determines the preference for glucose units versus alkyl moieties. E194 favors the binding of alkyl moieties, whereas K201 is more relevant for the binding of glucose units, in spite of its favorable interaction with alkyl moieties. The three residues E190, E194 and K201 reduce the affinity for phenyl moieties. In addition, M453 favors the binding of the second glucose unit of oligosaccharidic aglycones and also of the initial portion of alkyl-type aglycones. None of the residues investigated interacted with the terminal portion of alkyl-type aglycones. It was also demonstrated that E190, E194, K201 and M453 similarly contribute to stabilize ES(double dagger). Their interactions with aglycone are individually weaker than those formed by residues interacting with glycone, but their joint catalytic effects are similar. Finally, these interactions with aglycone do not influence glycone binding.     

Amino acid residues involved in substrate recognition of the Escherichia coli Orf135 protein

The Escherichia coli Orf135 protein, a MutT-type enzyme, hydrolyzes mutagenic 2-hydroxy-dATP (2-OH-dATP) and 8-hydroxy-dGTP, in addition to dCTP and 5-methyl-dCTP, and its deficiency causes increases in both the spontaneous and H(2)O(2)-induced mutation frequencies. To identify the amino acid residues that interact with these nucleotides, the Glu-33, Arg-72, Arg-77, and Asp-118 residues of Orf135, which are candidates for residues interacting with the base, were substituted, and the enzymatic activities of these mutant proteins were examined. The mutant proteins with a substitution at the 33rd, 72nd, and 118th amino acid residues displayed activities affected to various degrees for each substrate, suggesting the involvement of these residues in substrate binding. On the other hand, the mutant protein with a substitution at the 77th Arg residue had activitiy similar to that of the wild-type protein, excluding the possibility that this Arg side chain is involved in base recognition. In addition, the expression of some Orf135 mutants in orf135(-) E. coli reduced the level of formation of rpoB mutants elicited by H(2)O(2). These results reveal the residues involved in the substrate binding of the E. coli Orf135 protein.     

Amino acid residues in the nicotinamide binding site contribute to catalysis by horse liver alcohol dehydrogenase

Amino acid residues Thr-178, Val-203, and Val-292, which interact with the nicotinamide ring of the coenzyme bound to alcohol dehydrogenase (ADH), may facilitate hydride transfer and hydrogen tunneling by orientation and dynamic effects. The T178S, T178V, V203A, V292A, V292S, and V292T substitutions significantly alter the steady state and transient kinetics of the enzyme. The V292A, V292S, and V292T enzymes have decreased affinity for coenzyme (NAD+ by 30-50-fold and NADH by 35-75-fold) as compared to the wild-type enzyme. The substitutions in the nicotinamide binding site decrease the rate constant of hydride transfer for benzyl alcohol oxidation by 3-fold (for V292T ADH) to 16-fold (for V203A ADH). The modest effects suggest that catalysis does not depend critically on individual residues and that several residues in the nicotinamide binding site contribute to catalysis. The structures of the V292T ADH-NAD+-pyrazole and wild-type ADH-NAD+-4-iodopyrazole ternary complexes are very similar. Only subtle changes in the V292T enzyme cause the large changes in coenzyme binding and the small change in hydride transfer. In these complexes, one pyrazole nitrogen binds to the catalytic zinc, and the other nitrogen forms a partial covalent bond with C4 of the nicotinamide ring, which adopts a boat conformation that is postulated to be relevant for hydride transfer. The results provide an experimental basis for evaluating the contributions of dynamics to hydride transfer.     

Specific amino acid residues are involved in substrate discrimination and template binding of human REV1 protein

REV1 is a member of the Y-family DNA polymerases, but is atypical in utilizing only dCTP with a preference for guanine (G) as the template. Crystallography of the REV1-DNA-dCTP ternary complex has revealed a unique mechanism by which template G is evicted from the DNA helix and incoming dCTP is recognized by an arginine residue in an α-loop, termed the N-digit. To better understand functions of its individual amino acid residues, we made a series of mutant human REV1 proteins. We found that R357 and L358 play vital roles in template binding. Furthermore, extensive mutation analysis revealed a novel function of R357 for substrate discrimination, in addition to previously proposed specific interaction with incoming dCTP. We found that the binding pocket for dCTP of REV1 has also significant but latent affinity for dGTP. The results suggest that the positive charge on R357 could prevent interaction with dGTP. We propose that both direct and indirect mechanisms mediated by R357 ensure specificity for dCTP.     

Identification of carboxylic acid residues in glucoamylase G2 from Aspergillus niger that participate in catalysis and substrate binding

Functionally important carboxyl groups in glucoamylase G2 from Aspergillus niger were identified using a differential labelling approach which involved modification of the acarbose-inhibited enzyme with 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide (EAC) and inactivation by [3H]EAC following removal of acarbose. Subsequent sequence localization of the substituted acidic residues was facilitated by specific phenylthiohydantoins. The acid cluster Asp176, Glu179 and Glu180 reacted exclusively with [3H]EAC, while Asp112, Asp153, Glu259 and Glu389 had incorporated both [3H]EAC and EAC. It is conceivable that one or two of the [3H]EAC-labelled side chains act in catalysis while the other fully protected residue(s) participates in substrate binding probably together with the partially protected ones. Twelve carboxyl groups that reacted with EAC in the enzyme-acarbose complex were also identified. Asp176, Glu179 and Glu180 are all invariant in fungal glucoamylases. Glu180 was tentatively identified as a catalytic group on the basis of sequence alignments to catalytic regions in isomaltase and alpha-amylase. The partially radiolabelled Asp112 corresponds in Taka-amylase A to Tyr75 situated in a substrate binding loop at a distance from the site of cleavage. A possible correlation between carbodiimide modification of an essential carboxyl group and its role in the glucoamylase catalysis is discussed.     

Amino acid residues forming the active site of arylsulfatase A. Role in catalytic activity and substrate binding

Arylsulfatase A belongs to the sulfatase family whose members carry a Calpha-formylglycine that is post-translationally generated by oxidation of a conserved cysteine or serine residue. The formylglycine acts as an aldehyde hydrate with two geminal hydroxyls being involved in catalysis of sulfate ester cleavage. In arylsulfatase A and N-acetylgalactosamine 4-sulfatase this formylglycine was found to form the active site together with a divalent cation and a number of polar residues, tightly interconnected by a net of hydrogen bonds. Most of these putative active site residues are highly conserved among the eukaryotic and prokaryotic members of the sulfatase family. To analyze their function in binding and cleaving sulfate esters, we substituted a total of nine putative active site residues of human ASA by alanine (Asp29, Asp30, Asp281, Asn282, His125, His229, Lys123, Lys302, and Ser150). In addition the Mg2+-complexing residues (Asp29, Asp30, Asp281, and Asn282) were substituted conservatively by either asparagine or aspartate. In all mutants Vmax was decreased to 1-26% of wild type activity. The Km was more than 10-fold increased in K123A and K302A and up to 5-fold in the other mutants. In all mutants the pH optimum was increased from 4.5 by 0.2-0.8 units. These results indicate that each of the nine residues examined is critical for catalytic activity, Lys123 and Lys302 by binding the substrate and the others by direct (His125 and Asp281) or indirect participation in catalysis. The shift in the pH optimum is explained by two deprotonation steps that have been proposed for sulfate ester cleavage.     

Amino acid residues involved in stereoselective inhibition of cholinesterases with bambuterol

Bambuterol is a chiral carbamate known as selective inhibitor of butyrylcholinesterase (BChE). In order to relate bambuterol selectivity and stereoselectivity of cholinesterases to the active site residues, we studied the inhibition of recombinant mouse BChE, acetylcholinesterase (AChE) and six AChE mutants, employed to mimic BChE active site residues, by bambuterol enantiomers. Both enantiomers selectively inhibited BChE about 8000 times faster than AChE. The largest inhibition rate increase in comparison to AChE w.t. was observed with the F295L/Y337A mutant, showing that leucine 295 and alanine 337 are crucial residues in BChE for high bambuterol selectivity. All studied enzymes preferred inhibition by the R- over the S-bambuterol. The enlargement of the AChE choline binding site and of the acyl pocket by single or double mutations (Y337A, F295L/Y337A and F297I/Y337A) increased, in comparison to w.t. enzymes, inhibition rate constants of R- bambuterol more than that of S- bambuterol resulting in four times higher stereoselectivity. Peripheral site mutations (Y124Q and Y72N/Y124Q/Y337A) increased inhibition rate by S- more than R-bambuterol and consequently diminished the stereoselectivity.     

Mutagenesis identifies the critical amino acid residues of human endonuclease G involved in catalysis, magnesium coordination, and substrate specificity

Background  

Endonuclease G (EndoG), a member of DNA/RNA nonspecific ββα-Me-finger nucleases, is involved in apoptosis and normal cellular proliferation. In this study, we analyzed the critical amino acid residues of EndoG and proposed the catalytic mechanism of EndoG.     

Three residues involved in binding and catalysis in the carbamyl phosphate binding site of Escherichia coli aspartate transcarbamylase

Site-directed mutagenesis was used to create four mutant versions of Escherichia coli aspartate transcarbamylase at three positions in the catalytic chain of the enzyme. The location of all the amino acid substitutions was near the carbamyl phosphate binding site as previously determined by X-ray crystallography. Arg-54, which interacts with both the anhydride oxygen and a phosphate oxygen of carbamyl phosphate, was replaced by alanine. This mutant enzyme was approximately 17,000-fold less active than the wild type, although the binding of substrates and substrate analogues was not altered substantially. Arg-105, which interacts with both the carbonyl oxygen and a phosphate oxygen of carbamyl phosphate, was replaced by alanine. This mutant enzyme exhibited an approximate 1000-fold loss of activity, while the activity of catalytic subunit isolated from this mutant enzyme was reduced by 170-fold compared to the wild-type catalytic subunit. The KD of carbamyl phosphate and the inhibition constants for acetyl phosphate and N-(phosphono-acetyl)-L-aspartate (PALA) were increased substantially by this amino acid substitution. Furthermore, this loss in substrate and substrate analogue binding can be correlated with the large increases in the aspartate and carbamyl phosphate concentrations at half of the maximum observed specific activity, [S]0.5. Gln-137, which interacts with the amino group of carbamyl phosphate, was replaced by both asparagine and alanine. The asparagine mutant exhibited only a small reduction in activity while the alanine mutant was approximately 50-fold less active than the wild type. The catalytic subunits of both these mutant enzymes were substantially more active than the corresponding holoenzymes.(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of arginine residues in substrate binding and catalysis by deacetoxycephalosporin C synthase.

Deacetoxycephalosporin C synthase (DAOCS) catalyses the oxidative ring expansion of penicillin N, the committed step in the biosynthesis of cephamycin C by Streptomyces clavuligerus. Site-directed mutagenesis was used to investigate the seven Arg residues for activity (74, 75, 160, 162, 266, 306 and 307), selected on the basis of the DAOCS crystal structure. Greater than 95% of activity was lost upon mutation of Arg-160 and Arg266 to glutamine or other residues. These results are consistent with the proposed roles for these residues in binding the carboxylate linked to the nucleus of penicillin N (Arg160 and Arg162) and the carboxylate of the alpha-aminoadipoyl side-chain (Arg266). The results for mutation of Arg74 and Arg75 indicate that these residues play a less important role in catalysis/binding. Together with previous work, the mutation results for Arg306 and Arg307 indicate that modification of the C-terminus may be profitable with respect to altering the penicillin side-chain selectivity of DAOCS.     

Amino acid residues in the GIY-YIG endonuclease II of phage T4 affecting sequence recognition and binding as well as catalysis

Phage T4 endonuclease II (EndoII), a GIY-YIG endonuclease lacking a carboxy-terminal DNA-binding domain, was subjected to site-directed mutagenesis to investigate roles of individual amino acids in substrate recognition, binding, and catalysis. The structure of EndoII was modeled on that of UvrC. We found catalytic roles for residues in the putative catalytic surface (G49, R57, E118, and N130) similar to those described for I-TevI and UvrC; in addition, these residues were found to be important for substrate recognition and binding. The conserved glycine (G49) and arginine (R57) were essential for normal sequence recognition. Our results are in agreement with a role for these residues in forming the DNA-binding surface and exposing the substrate scissile bond at the active site. The conserved asparagine (N130) and an adjacent proline (P127) likely contribute to positioning the catalytic domain correctly. Enzymes in the EndoII subfamily of GIY-YIG endonucleases share a strongly conserved middle region (MR, residues 72 to 93, likely helical and possibly substituting for heterologous helices in I-TevI and UvrC) and a less strongly conserved N-terminal region (residues 12 to 24). Most of the conserved residues in these two regions appeared to contribute to binding strength without affecting the mode of substrate binding at the catalytic surface. EndoII K76, part of a conserved NUMOD3 DNA-binding motif of homing endonucleases found to overlap the MR, affected both sequence recognition and catalysis, suggesting a more direct involvement in positioning the substrate. Our data thus suggest roles for the MR and residues conserved in GIY-YIG enzymes in recognizing and binding the substrate.     

Amino acid residues involved in organic solvent-stability of the LST-03 lipase

LST-03 lipase from Pseudomonas aeruginosa LST-03 is highly active and stable in the presence of various organic solvents. To further characterize and improve the organic solvent-stability of the LST-03 lipase, residues that potentially provide this stability were identified and mutated to other amino acids in an effort to increase the organic solvent-stability of the protein. S155L, G157R, S164K, S194R, and D209N mutations were found to improve the organic solvent-stability of the wild-type LST-03 lipase. Such mutations were found to induce structural changes, including the formation of a salt bridge, hydrogen bonds, lead to an improved packing of the hydrophobic core, and pI shift of side chain. These changes increased the stability of the protein, thereby improving the organic solvent-stability of the wild-type LST-03 lipase. In addition, a single mutation was found to stabilize the lipase by single or multiple factors.     

Conserved and nonconserved residues in the substrate binding site of 7,8-diaminopelargonic acid synthase from Escherichia coli are essential for catalysis

The vitamin B(6)-dependent enzyme 7,8-diaminopelargonic acid (DAPA) synthase catalyzes the antepenultimate step in the synthesis of biotin, the transfer of the alpha-amino group of S-adenosyl-l-methionine (SAM) to 7-keto-8-aminopelargonic acid (KAPA) to form DAPA. The Y17F, Y144F, and D147N mutations in the active site were constructed independently. The k(max)/K(m)(app) values for the half-reaction with DAPA of the Y17F and Y144F mutants are reduced by 1300- and 2900-fold, respectively, compared to the WT enzyme. Crystallographic analyses of these mutants do not show significant changes in the structure of the active site. The kinetic deficiencies, together with a structural model of the enzyme-PLP/DAPA Michaelis complex, point to a role of these two residues in recognition of the DAPA/KAPA substrates and in catalysis. The k(max)/K(m)(app) values for the half-reaction with SAM are similar to that of the WT enzyme, showing that the two tyrosine residues are not involved in this half-reaction. Mutations of the conserved Arg253 uniquely affect the SAM kinetics, thus establishing this position as part of the SAM binding site. The D147N mutant is catalytically inactive in both half-reactions. The structure of this mutant exhibits significant changes in the active site, indicating that this residue plays an important structural role. Of the four residues examined, only Tyr144 and Arg253 are strictly conserved in the available amino acid sequences of DAPA synthases. This enzyme thus provides an illustrative example that active site residues essential for catalysis are not necessarily conserved, i.e., that during evolution alternative solutions for efficient catalysis by the same enzyme arose. Decarboxylated SAM [S-adenosyl-(5')-3-methylthiopropylamine] reacts nearly as well as SAM and cannot be eliminated as a putative in vivo amino donor.     

Amino acid residues essential for catalysis by peptidyl dipeptidase-4 from Pseudomonas maltophilia

To assess residues essential for catalysis by prokaryotic peptidyl dipeptidase-4, the enzyme was subjected to chemical modification by a series of reagents. Treatment with either tetranitromethane or N-acetylimidazole abolished catalytic activity. Hydroxylamine reversed inactivation by acetylimidazole only. Thus, an essential tyrosine is indicated. Enzymatic activity also was quenched by either trinitrobenzenesulfonic acid or diethyl pyrocarbonate. Inactivation by these reagents was not reversed by hydroxylamine. These data suggest an essential lysine. The competitive inhibitor Phe-Arg protected partially against inactivation by tetranitromethane, and fully against inactivation by N-acetylimidazole. The substrate Hip-Phe-Arg protected against inactivation by trinitrobenzenesulfonic acid and diethyl pyrocarbonate. Thus, both tyrosine and lysine are located at the catalytic site.     

Amino acid residues 62 and 193 play the key role in regulating the synergism of substrate binding in oyster arginine kinase

The purpose of this study is to clarify the amino acid residues responsible for the synergism in substrate binding of arginine kinase (AK), a key enzyme in invertebrate energy metabolism. AKs contain a pair of highly conserved amino acids (D62 and R193) that form an ion pair, and replacement of these residues can cause a pronounced loss of activity. Interestingly, in the oyster Crassostrea AK, these residues are replaced by an N and a K, respectively. Despite this replacement, the enzyme retains high activity and moderate synergism in substrate binding (Kd/Km=2.3). We replaced the N62 by G or D and the K193 by G or R in Crassostrea AK, and also constructed the double mutants of N62G/K193G and N62D/K193R. All of the mutants retained 50-90% of the wild-type activity. In N62G and N62D mutants, the Kmarg for arginine binding was comparable to that of wild-type enzyme, but the Kdarg was increased 2-5-fold, resulting in a strong synergism (Kd/Km=4.9-11.3). On the other hand, in K193G and K193R mutants, the Kmarg was increased 4-fold, and synergism was lost almost completely (Kd/Km=1.0-1.4). The N62G/K193G double mutant showed similar characteristics to the K193G and K193R mutants. Another double mutant, N62D/K193R, similar to the amino acid pair in the wild-type enzyme, had characteristics similar to those of the wild-type enzyme. These results indicate that the amino acid residues 62 and 193 play the key role in mediating the synergism in substrate binding of oyster arginine kinase.     

Site-directed mutagenesis of 2,4-dichlorophenoxyacetic acid/alpha-ketoglutarate dioxygenase. Identification of residues involved in metallocenter formation and substrate binding

2,4-dichlorophenoxyacetic acid (2,4-D)/alpha-ketoglutarate (alpha-KG) dioxygenase (TfdA) is an Fe(II)-dependent enzyme that catalyzes the first step in degradation of the herbicide 2,4-D. The active site structures of a small number of enzymes within the alpha-KG-dependent dioxygenase superfamily have been characterized and shown to have a similar HXDX(50-70)HX(10)RXS arrangement of residues that make up the binding sites for Fe(II) and alpha-KG. TfdA does not have obvious homology to the dioxygenases containing the above motif but is related in sequence to eight other enzymes in the superfamily that form a distinct consensus sequence (HX(D/E)X(138-207) HX(10)R/K). Variants of TfdA were created to examine the roles of putative metal-binding residues and the functions of the other seven histidines in this protein. The H167A, H200A, H213A, H245A, and H262A forms of TfdA formed inclusion bodies when overproduced in Escherichia coli DH5alpha; however, these proteins were soluble when fused to the maltose-binding protein (MBP). MBP-TfdA exhibited kinetic parameters similar to the native enzyme. The H8A and H235A variants were catalytically similar to wild-type TfdA. MBP-H213A and H216A TfdA have elevated K(m) values for 2,4-D, and the former showed a decreased k(cat), suggesting these residues may affect substrate binding or catalysis. The H113A, D115A, MBP-H167A, MBP-H200A, MBP-H245A and MBP-H262A variants of TfdA were inactive. Gel filtration analysis revealed that the latter two proteins were highly aggregated. The remaining four inactive variants were examined in their Cu(II)-substituted forms by EPR and electron spin-echo envelope modulation (ESEEM) spectroscopic methods. Changes in EPR spectra upon addition of substrates indicated that copper was present at the active site in the H113A and D115A variants. ESEEM analysis revealed that two histidines are bound equatorially to the copper in the D115A and MBP-H167A TfdA variants. The experimental data and sequence analysis lead us to conclude that His-113, Asp-115, and His-262 are likely metal ligands in TfdA and that His-213 may aid in catalysis or binding of 2,4-D.     

Identification of thromboxane synthase amino acid residues involved in heme-propionate binding

Thromboxane A2 synthase (TXAS) is a member of the cytochrome P450 superfamily and catalyzes an isomerization reaction that converts prostaglandin H2 to thromboxane A2. As a step toward understanding the structure/function relationships of TXAS, we mutated amino acid residues predicted to bind the propionate groups of A- and D-pyrrole rings of the heme. These mutations at each of these residues (Asn-110, Trp-133, Arg-137, Arg-413, and Arg-478) resulted in altered heme binding, as evidenced by perturbation of the absorption spectra and EPR. The mutations, although causing no significant changes in the secondary structure of the proteins, induced tertiary structural changes that led to increased susceptibility to trypsin digestion and alteration of the intrinsic protein fluorescence. Moreover, these mutant proteins lost their binding affinity to the substrate analog, had a lower heme content and retained less than 5% of the wild-type catalytic activity. However, mutations at the neighboring amino acid of the aforementioned residues yielded mutant proteins retaining the biochemical and biophysical properties of the wild type TXAS. Aligning the TXAS sequence with the structurally known P450s, we proposed that in TXAS the A-ring propionate of the heme is hydrogen bonded to Asn-110, Arg-413, and Arg-478, whereas D-ring propionate is hydrogen bonded to Trp-133 and Arg-137. Furthermore, both A- and D-ring propionates bulge away from the heme plane and both lie on the proximal face of heme plane, a structure similar to P450terp.     

Involvement of conserved aspartate and glutamate residues in the catalysis and substrate binding of maize starch synthase

Chemical modification of maize starch synthase IIb-2 (SSIIb-2) using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC), which modifies acidic amino acid residues, resulted in a time- and concentration-dependent inactivation of SSIIb-2. ADPGlc was found to completely protect SSIIb-2 from inactivation by EDAC. These results suggest that glutamate or aspartate is important for SS activity. On the basis of the sequence identity of SS, conserved acidic amino acids were mutagenized to identify the specific amino acid residues important for SS activity. Three amino acids (D21, D139, and E391) were found to be important for SS activity. D21N showed 4% of the wild-type enzyme activity and a 10-fold decrease in the affinity for ADPGlc, while the conservative change from D21 to E resulted in a decrease in V(max) and no change in affinity for ADPGlc, suggesting that the negative charge is important for ADPGlc binding. When sites D139 and E391 were changed to their respective amide form, no SS activity was detected. With the conservative change, D139E showed a decrease in V(max) and no changes in apparent K(m) for substrates. E391D showed a 9-fold increase in K(m) for ADPGlc, a 12-fold increase in apparent K(m) for glycogen, and a 4-fold increase in apparent K(m) for amylopectin. The circular dichroism analysis indicates that these kinetic changes may not be due to a major conformation change in the protein. These results provide the first evidence that the conserved aspartate and glutamate residues could be involved in the catalysis or substrate binding of SS.