Identification of the enzymatic active site of CD38 by site-directed mutagenesis

CD38 is a ubiquitous protein originally identified as a lymphocyte antigen and recently also found to be a multifunctional enzyme participating in the synthesis and metabolism of two Ca(2+) messengers, cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate. It is homologous to Aplysia ADP-ribosyl cyclase, where the crystal structure has been determined. Residues of CD38 corresponding to those at the active site of the Aplysia cyclase were mutagenized. Changing Glu-226, which corresponded to the catalytic residue of the cyclase, to Asp, Asn, Gln, Leu, or Gly eliminated essentially all enzymatic activities of CD38, indicating it is most likely the catalytic residue. Photoaffinity labeling showed that E226G, nevertheless, retained substantial NAD binding activity. The secondary structures of these inactive mutants as measured by circular dichroism were essentially unperturbed as compared with the wild type. Other nearby residues were also investigated. The mutants D147V and E146L showed 7- and 19-fold reduction in NADase activity, respectively. The cADPR hydrolase activity of the two mutants was similarly reduced. Asp-155, on the other hand, was crucial for the GDP-ribosyl cyclase activity since its substitution with either Glu, Asn, or Gln stimulated the activity 3-15-fold, whereas other activities remained essentially unchanged. In addition to these acidic residues, two tryptophans were also important, since all enzyme activities of W125F, W125Y, W189G and W189Y were substantially reduced. This is consistent with the two tryptophans serving a substrate positioning function. A good correlation was observed when the NADase activity of all the mutants was plotted against the cADPR hydrolase activity. Homology modeling revealed all these critical residues are clustered in a pocket near the center of the CD38 molecule. The results indicate a strong structural homology between the active sites of CD38 and the Aplysia cyclase.     

Probing the phosphocholine-binding site of human C-reactive protein by site-directed mutagenesis.

Human C-reactive protein (CRP) can activate the classical pathway of complement and function as an opsonin only when it is complexed to an appropriate ligand. Most known CRP ligands bind to the phosphocholine (PCh)-binding site of the protein. In the present study, we used oligonucleotide-directed site-specific mutagenesis to investigate structural determinants of the PCh-binding site of CRP. Eight mutant recombinant (r) CRP, Y40F; E42Q; Y40F, E42Q; K57Q; R58G; K57Q, R58G; W67K; and K57Q, R58G, W67K were constructed and expressed in COS cells. Wild-type and all mutant rCRP except for the W67K mutants bound to solid-phase PCh-substituted bovine serum albumin (PCh-BSA) with similar apparent avidities. However, W67K rCRP had decreased avidity for PCh-BSA and the triple mutant, K57Q, R58G, W67K, failed to bind PCh-BSA. Inhibition experiments using PCh and dAMP as inhibitors indicated that both Lys-57 and Arg-58 contribute to PCh binding. They also indicated that Trp-67 provides interactions with the choline group. The Y40F and E42Q mutants were found to have increased avidity for fibronectin compared to wild-type rCRP. We conclude that the residues Lys-57, Arg-58, and Trp-67 contribute to the structure of the PCh-binding site of human CRP. Residues Tyr-40 and Glu-42 do not appear to participate in the formation of the PCh-binding site of CRP, however, they may be located in the vicinity of the fibronectin-binding site of CRP.     

Exposing a hidden functional site of C-reactive protein by site-directed mutagenesis

C-reactive protein (CRP) is a cyclic pentameric protein whose major binding specificity, at physiological pH, is for substances bearing exposed phosphocholine moieties. Another pentameric form of CRP, which exists at acidic pH, displays binding activity for oxidized LDL (ox-LDL). The ox-LDL-binding site in CRP, which is hidden at physiological pH, is exposed by acidic pH-induced structural changes in pentameric CRP. The aim of this study was to expose the hidden ox-LDL-binding site of CRP by site-directed mutagenesis and to generate a CRP mutant that can bind to ox-LDL without the requirement of acidic pH. Mutation of Glu(42), an amino acid that participates in intersubunit interactions in the CRP pentamer and is buried, to Gln resulted in a CRP mutant (E42Q) that showed significant binding activity for ox-LDL at physiological pH. For maximal binding to ox-LDL, E42Q CRP required a pH much less acidic than that required by wild-type CRP. At any given pH, E42Q CRP was more efficient than wild-type CRP in binding to ox-LDL. Like wild-type CRP, E42Q CRP remained pentameric at acidic pH. Also, E42Q CRP was more efficient than wild-type CRP in binding to several other deposited, conformationally altered proteins. The E42Q CRP mutant provides a tool to investigate the functions of CRP in defined animal models of inflammatory diseases including atherosclerosis because wild-type CRP requires acidic pH to bind to deposited, conformationally altered proteins, including ox-LDL, and available animal models may not have sufficient acidosis or other possible modifiers of the pentameric structure of CRP at the sites of inflammation.     

Pre-steady state transport by erythrocyte band 3 protein: uphill countertransport induced by the impermeant inhibitor H2DIDS.

Pre-steady state Cl- efflux experiments have been performed to test directly the idea that the transport inhibitor H2DIDS (4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonate) binds preferentially to the outward-facing state of the transporter. Cells were equilibrated with a medium consisting of 150 mM sodium phosphate, pH 6.2, N2 atmosphere, and 80-250 microM 36Cl-. Addition of H2DIDS (10-fold molar excess compared with band 3) induces a transient efflux of Cl-, as expected if H2DIDS binds more tightly to outward-facing than to inward-facing states. The size of the H2DIDS-induced efflux depends on the Cl- concentration and is about 700,000 ions per cell at the highest concentrations tested. The size of the transient efflux is larger than would be expected if the catalytic cycle for anion exchange involved one pair of exchanging anions per band 3 dimer. These results are completely consistent with a ping-pong mechanism of anion exchange in which the catalytic cycle consists of one pair of exchanging anions per subunit of the band 3 dimer.     

Function of Lys-166 of Rhodospirillum rubrum ribulosebisphosphate carboxylase/oxygenase as examined by site-directed mutagenesis

Affinity labeling and comparative sequence analyses have placed Lys-166 of ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum at the active site. The unusual nucleophilicity and acidity of the epsilon-amino group of Lys 166 (pKa = 7.9) suggest its involvement in catalysis, perhaps as the base that enolizes ribulosebisphosphate (Hartman, F.C., Milanez, S., and Lee, E.H. (1985) J. Biol. Chem. 260, 13968-13975). In attempts to clarify the role of Lys-166 of the carboxylase, we have used site-directed mutagenesis to replace this lysyl residue with glycine, alanine, serine, glutamine, arginine, cysteine, or histidine. All seven of these mutant proteins, purified by immunoaffinity chromatography, are severely deficient in carboxylase activity; the serine mutant, which is the most active, has a kcat only 0.2% that of the wild-type enzyme. Although low, the carboxylase activity displayed by some of the mutant proteins proves that Lys-166 is not required for substrate binding and argues that the detrimental effects brought about by amino acid substitutions at position 166 do not reflect gross conformational changes. As demonstrated by their ability to tightly bind a transition-state analogue (2-carboxyarabinitol 1,5-bisphosphate) in the presence of CO2 and Mg2+, some of the mutant proteins undergo the carbamylation reaction that is required for activation of the wild-type enzyme. Since Lys-166 is required neither for activation (i.e. carbamylation by CO2) nor for substrate binding, it must be essential to catalysis. When viewed within the context of previous related studies, the results of site-directed mutagenesis are entirely consistent with Lys-166 functioning as the base that initiates catalysis by abstracting the C-3 proton from ribulosebisphosphate. An alternative possibility that Lys-166 acts to stabilize a transition state in the reaction pathway cannot be rigorously excluded.     

Stabilization of penicillin G acylase from Escherichia coli: site-directed mutagenesis of the protein surface to increase multipoint covalent attachment

Three mutations on the penicillin acylase surface (increasing the number of Lys in a defined area) were performed. They did not alter the enzyme's stability and kinetic properties; however, after immobilization on glyoxyl-agarose, the mutant enzyme showed improved stability under all tested conditions (e.g., pH 2.5 at 4 degrees C, pH 5 at 60 degrees C, pH 7 at 55 degrees C, or 60% dimethylformamide), with stabilization factors ranging from 4 to 11 compared with the native enzyme immobilized on glyoxyl-agarose.     

Comparison of covalent with reversible inhibitor binding sites of the gastric H,K-ATPase by site-directed mutagenesis

The gastric H,K-ATPase is covalently inhibited by substituted pyridyl-methylsulfinyl-benzimidazoles, such as omeprazole, that convert to thiophilic probes of luminally accessible cysteines in the acid space. The K(+) competitive inhibitor, SCH28080, prevented inhibition of acid transport by omeprazole. In stably expressing HEK293 cells, the benzimidazole-reactive cysteines, Cys-321 (transmembrane helix (TM) 3), Cys-813 and Cys-822 (TM5/6), and Cys-892 (TM7/8) were mutated to the amino acids found in the SCH28080-resistant Na,K-ATPase and kinetic parameters of H,K-ATPase activity analyzed. Mutations of Cys-822 and Cys-892 had insignificant effects on the K(i(app)), K(m(app)) or V(max), but mutations of Cys-813 to threonine and Cys-321 to alanine decreased the affinity for SCH28080. Mutation of Cys-321 to alanine produced mixed kinetics of inhibition, still with higher affinity for the cation-free form of phosphoenzyme. Since the phenylmethoxy ring of the imidazo-pyridine inhibitors binds to TM1/2, as shown by earlier photoaffinity studies, and the mutations in TM6 (Cys-813 --> Thr) as well as the end of TM3 (Cys-321 --> Ala) decrease the affinity for SCH28080, the TM1/2, TM3, and TM6 helices lie within approximately 16 A of each other based on the size of the active, extended conformation of SCH28080.     

Probing the ubiquinol-binding site in cytochrome bd by site-directed mutagenesis

To probe the structure of the quinol oxidation site in loop VI/VII of the Escherichia coli cytochrome bd, we substituted three conserved residues (Gln249, Lys252, and Glu257) in the N-terminal region and three glutamates (Glu278, Glu279, and Glu280) in the first internal repeat. We found that substitutions of Glu257 by Ala or Gln, and Glu279 and Glu280 by Gln, severely reduced the oxidase activity and the expression level of cytochrome bd. In contrast, Lys252 mutations reduced only the oxidase activity. Blue shifts in the 440 and 630 nm peaks of the reduced Lys252 mutants and in the 561 nm peak of the reduced Glu257 mutants indicate the proximity of Lys252 to the heme b(595)-d binuclear center and Glu257 to heme b(558), respectively. Perturbations of reduced heme b(558) upon binding of aurachin D support structural changes in the quinol-binding site of the mutants. Substitutions of Lys252 and Glu257 caused large changes in kinetic parameters for the ubiquinol-1 oxidation. These results indicate that Lys252 and Glu257 in the N-terminal region of the Q-loop are involved in the quinol oxidation by bd-type terminal oxidase.     

Identification of the enzymatic active site of tobacco caffeoyl-coenzyme A O-methyltransferase by site-directed mutagenesis.

Animal catechol O-methyltransferases and plant caffeoyl-coenzyme A O-methyltransferases share about 20% sequence identity and display common structural features. The crystallographic structure of rat liver catechol O-methyltransferase was used as a template to construct a homology model for tobacco caffeoyl-coenzyme A O-methyltransferase. Integrating substrate specificity data, the three-dimensional model identified several amino acid residues putatively involved in substrate binding. These residues were mutated by a polymerase chain reaction method and wild-type and mutant enzymes were each expressed in Escherichia coli and purified. Substitution of Arg-220 with Thr resulted in the total loss of enzyme activity, thus indicating that Arg-220 is involved in the electrostatic interaction with the coenzyme A moiety of the substrate. Changes of Asp-58 to Ala and Gln-61 to Ser were shown to increase K(m) values for caffeoyl coenzyme A and to decrease catalytic activity. Deletions of two amino acid sequences specific for plant enzymes abolished activity. The secondary structures of the mutants, as measured by circular dichroism, were essentially unperturbed as compared with the wild type. Similar changes in circular dichroism spectra were observed after addition of caffeoyl coenzyme A to the wild-type enzyme and the substitution mutants but not in the case of deletion mutants, thus revealing the importance of these sequences in substrate-enzyme interactions.     

Methionine 500, the site of covalent attachment of an active site-directed reagent of beta-galactosidase.

The site of attachment to beta-galactosidase of the active site-directed inhibitor, beta-D-galactopyranosylmethyl p-nitrophenyl triazene, was determined. When the enzyme is completely inactivated, 1 mol of the galactopyranosylmethyl group is bound per mol of monomer with retention of the tetrameric structure. After reaction with the [14C]methyl reagent, labeled peptides were isolated and analyzed. The radioactive label was found to be covalently bound to methionine residue 500.     

Identification of active site residues of Escherichia coli fumarate reductase by site-directed mutagenesis.

Menaquinol-fumarate oxidoreductase of Escherichia coli is a four-subunit membrane-bound complex that catalyzes the final step in anaerobic respiration when fumarate is the terminal electron acceptor. The enzyme is structurally and catalytically similar to succinate dehydrogenase (succinate-ubiquinone oxidoreductase) from both procaryotes and eucaryotes. Both enzymes have been proposed to contain an essential cysteine residue at the active site based on studies with thiol-specific reagents. Chemical modification studies have also suggested roles for essential histidine and arginine residues in catalysis by succinate dehydrogenase. In the present study, a combination of site-directed mutagenesis and chemical modification techniques have been used to investigate the role(s) of the conserved histidine 232, cysteine 247, and arginine 248 residues of the flavorprotein subunit (FrdA) in active site function. A role for His-232 and Arg-248 of FrdA is shown by loss of both fumarate reductase and succino-oxidase activities following site-directed substitution of these particular amino acids. Evidence is also presented that suggests a second arginine residue may form part of the active site. Potential catalytic and substrate-binding roles for arginine are discussed. The effects of removing histidine-232 of FrdA are consistent with its proposed role as a general acid-base catalyst. The fact that succinate oxidation but not fumarate reduction was completely lost, however, might suggest that alternate proton donors substitute for His-232. The data confirm that cysteine 247 of FrdA is responsible for the N-ethylmaleimide sensitivity shown by fumarate reductase but is not required for catalytic activity or the tight-binding of oxalacetate, as previously thought.     

Exploring the active site of plant glutaredoxin by site-directed mutagenesis

Six mutants (Y26A, C27S, Y29F, Y29P, C30S and Y26W/Y29P) have been engineered in order to explore the active site of poplar glutaredoxin (Grx) (Y26CPYC30). The cysteinic mutants indicate that Cys 27 is the primary nucleophile. Phe is a good substitute for Tyr 29, but the Y29P mutant was inactive. The Y26A mutation caused a moderate loss of activity. The YCPPC and WCPPC mutations did not improve the reactivity of Grx with the chloroplastic NADP-malate dehydrogenase, a well known target of thioredoxins (Trxs). The results are discussed in relation with the known biochemical properties of Grx and Trx.     

Identification of the active site residues in dipeptidyl peptidase IV by affinity labeling and site-directed mutagenesis.

The active site of dipeptidyl peptidase IV (DPPIV) was examined by chemical modification and site-directed mutagenesis. Purified DPPIV was covalently modified with [3H]diisopropyl fluorophosphate (DFP). The radiolabeled DPPIV was digested with lysyl endopeptidase, and the peptides were separated by high-performance liquid chromatography. A single 3H-containing peptide was obtained and analyzed for amino acid sequence and radioactivity distribution. A comparison of the determined sequence with the predicted primary structure of DPPIV [Ogata, S., Misumi, Y., & Ikehara, Y. (1989) J. Biol. Chem. 264, 3596-3601] revealed that [3H]DFP was bound to Ser631 within the sequence Gly629-Trp-Ser-Tyr-Gly633, which corresponds to the consensus sequence Gly-X-Ser-X-Gly proposed for serine proteases. To further identify the essential residues in the active-site sequence, we modified the DPPIV cDNA by site-directed mutagenesis to encode its variants. Expression of the mutagenized cDNAs in COS-1 cells demonstrated that any single substitution of Gly629, Ser631, or Gly633 with other residues resulted in the complete loss of the enzyme activity and DFP binding. Although substitution of Trp630----Glu or Tyr632----Phe caused no effect on the enzyme activity, that of Tyr632----Leu or Gly abolished the activity. These results indicate that the sequence Gly-X-Ser-(Tyr)-Gly is essential for the expression of the DPPIV activity.     

Identification of putative residues involved in the accessibility of the substrate-binding site of lipoxygenase by site-directed mutagenesis studies

Lipoxygenases (LOXs) are a class of widespread dioxygenases catalyzing the hydroperoxidation of polyunsaturated fatty acids (PUFA). Recently, we isolated a cDNA encoding a LOX, named olive LOX1, from olive fruit of which the deduced amino acid sequence shows more than 50% identity with plant LOXs. In the present study, a model of olive LOX1 based on the crystal structure of soybean LOX-1 as template has been generated and two bulky amino acid residues highly conserved in LOXs (Phe277) and in plant LOXs (Tyr280), located at the putative entrance of catalytic site were identified. These residues may perturb accessibility of the substrate-binding site and therefore were substituted by less space-filling residues. Kinetic studies using linoleic and linolenic acids as substrates were carried out on wild type and mutants. The results show that the removal of steric bulk at the entrance of the catalytic site induces an increase of substrate affinity and of catalytic efficiency, and demonstrate that penetration of substrates into active site of olive LOX1 requires the movement of the side chains of the Phe277 and Tyr280 residues. This study suggests the involvement of these residues in the accessibility of the substrate-binding site in the lipoxygenase family.     

Essentiality of Lys-329 of ribulose-1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum as demonstrated by site-directed mutagenesis

The unusual chemical properties of active-site Lys-329 of ribulose bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum have suggested that this residue is required for catalysis. To test this postulate Lys-329 was replaced with glycine, serine, alanine, cysteine, arginine, glutamic acid or glutamine by site-directed mutagenesis. These single amino acid substitutions do not appear to induce major conformational changes because (i) intersubunit interactions are unperturbed in that the purified mutant proteins are stable dimers like the wild-type enzyme and (ii) intrasubunit folding is normal in that the mutant proteins bind the competitive inhibitor 6-phosphogluconate with an affinity similar to that of wild-type enzyme. In contrast, all of the mutant proteins are severely deficient in carboxylase activity (less than 0.01% of wild-type) and are unable to form the exchange-inert complex, characteristic of the wild-type enzyme, with the transition-state analogue carboxyarabinitol bisphosphate. These results underscore the stringency of the requirement for a lysyl side-chain at position 329 and imply that Lys-329 is involved in catalysis, perhaps stabilizing a transition state in the overall reaction pathway.     

Glutamate synthase: identification of the NADPH-binding site by site-directed mutagenesis

To contribute to the understanding of glutamate synthase and of beta subunit-like proteins, which have been detected by sequence analyses, we identified the NADPH-binding site out of the two potential ADP-binding regions found in the beta subunit. The substitution of an alanyl residue for G298 of the beta subunit of Azospirillum brasilense glutamate synthase (the second glycine in the GXGXXA fingerprint of the postulated NADPH-binding site) yielded a protein species in which the flavin environment and properties are unaltered. On the contrary, the binding of the pyridine nucleotide substrate is significantly perturbed demonstrating that the C-terminal potential ADP-binding fold of the beta subunit is indeed the NADPH-binding site of the enzyme. The major effect of the G298A substitution in the GltS beta subunit consists of an approximately 10-fold decrease of the affinity of the enzyme for pyridine nucleotides with little or no effect on the rate of the enzyme reduction by NADPH. By combining kinetic measurements and absorbance-monitored equilibrium titrations of the G298A-beta subunit mutant, we conclude that also the positioning of its nicotinamide portion into the active site is altered thus preventing the formation of a stable charge-transfer complex between reduced FAD and NADP(+). During the course of this work, the Azospirillum DNA regions flanking the gltD and gltB genes, the genes encoding the GltS beta and alpha subunits, respectively, were sequenced and analyzed. Although the Azospirillum GltS is similar to the enzyme of other bacteria, it appears that the corresponding genes differ with respect to their arrangement in the chromosome and to the composition of the glt operon: no genes corresponding to E. coli and Klebsiella aerogenes gltF or to Bacillus subtilis gltC, encoding regulatory proteins, are found in the DNA regions adjacent to that containing gltD and gltB genes in Azospirillum. Further studies are needed to determine if these findings also imply differences in the regulation of the glt genes expression in Azospirillum (a nitrogen-fixing bacterium) with respect to enteric bacteria.     

Role of the amino acid invariants in the active site of MurG as evaluated by site-directed mutagenesis

To evaluate their role in the active site of the MurG enzyme from Escherichia coli, 13 residues conserved in the sequences of 73 MurG orthologues were submitted to site-directed mutagenesis. All these residues lay within, or close to, the active site of MurG as defined by its tridimensional structure [Ha et al., Prot. Sci. 9 (2000) 1045-1052, and Hu et al., Proc. Natl. Acad. Sci. USA 100 (2003) 845-849]. Thirteen mutants proteins, in which residues T15, H18, Y105, H124, E125, N127, N134, S191, N198, R260, E268, Q288 or N291 have been replaced by alanine, were obtained as the C-terminal His-tagged forms. The effects of the mutations on the activity were checked: (i) by functional complementation of an E. coli murG mutant strain by the mutated genes; and (ii) by the determination of the steady-state kinetic parameters of the purified proteins. Most mutations resulted in an important loss of activity and, in the case of N134A, in the production of a highly unstable protein. The results correlated with the assigned or putative functions of the residues based on the tridimensional structure.