Identification by site-directed mutagenesis of a hydrophobic binding site of the mitochondrial carnitine/acylcarnitine carrier involved in the interaction with acyl groups

The role of hydrophobic residues of the mitochondrial carnitine/acylcarnitine carrier (CAC) in the inhibition by acylcarnitines has been investigated by site-directed mutagenesis. According to the homology model of CAC in cytosolic opened conformation (c-state), L14, G17, G21, V25, P78, V82, M85, C89, F93, A276, A279, C283, F287 are located in the 1st (H1), 2nd (H2) and 6th (H6) transmembrane α-helices and exposed in the central cavity, forming a hydrophobic half shell. These residues have been substituted with A (or G) and in some cases with M. Mutants have been assayed for transport activity measured as [(3)H]carnitine/carnitine antiport in proteoliposomes. With the exception of G17A and G21M, mutants exhibited activity from 20% to 100% of WT. Among the active mutants only G21A, V25M, P78A and P78M showed Vmax lower than half and/or Km more than two fold respect to WT. Acylcarnitines competitively inhibited carnitine antiport. The extent of inhibition of the mutants by acylcarnitines with acyl chain length of 2, 4, 8, 12, 14 and 16 has been compared with the WT. V25A, P78A, P78M and A279G showed reduced extent of inhibition by all the acylcarnitines; V25M showed reduced inhibition by shorter acylcarnitines; V82A, V82M, M85A, C89A and A276G showed reduced inhibition by longer acylcarnitines, respect to WT. C283A showed increased extent of inhibition by acylcarnitines. Variations of Ki of mutants for acylcarnitines reflected variations of the inhibition profiles. The data demonstrated that V25, P78, V82, M85 and C89 are involved in the acyl chain binding to the CAC in c-state.     

Site-directed mutagenesis and chemical modification of the six native cysteine residues of the rat mitochondrial carnitine carrier: implications for the role of cysteine-136

By use of site-directed mutagenesis in combination with chemical modification of mutated proteins, the role of the six Cys residues in the transport function of the rat mitochondrial carnitine carrier (CAC) was studied. Several CAC mutants, in which one or more Cys residues had been replaced with Ser, were overexpressed in Escherichia coli, purified, and reconstituted in liposomes. The efficiency of incorporation into liposomes of the reconstituted proteins was lower for all constructs lacking Cys-23. Single, double, and quadruple replacement mutants showed V(max) comparable to that of the wild type. On the basis of the values of internal and external transport affinities (K(m)) for carnitine and of their comparison with those measured in mitochondria, the recombinant CAC is oriented unidirectionally in the liposomes, right side out compared to mitochondria. Substitution of Cys-136 with Ser caused a nearly complete loss of sensitivity of the CAC to N-ethylmaleimide, (2-aminoethyl)methanethiosulfonate hydrobromide (MTSES), and other hydrophilic SH reagents but not to the very hydrophobic N-phenylmaleimide. The wild-type CAC and the mutants containing Cys-136 showed substrate protection against NEM and MTSES inhibition and against NEM labeling. The data show that none of the native cysteines is essential for the transport mechanism and that Cys-136 is the major target of SH reagents and raise the hypothesis that Cys-136 is accessible from the external medium and is located at, or near, the substrate binding site. A model of the CAC is proposed in which the matrix hydrophilic loop containing Cys-136 protrudes into the membrane between the transmembrane domains of the protein.     

Post-translational modification by acetylation regulates the mitochondrial carnitine/acylcarnitine transport protein

The carnitine/acylcarnitine transporter (CACT; SLC25A20) mediates an antiport reaction allowing entry of acyl moieties in the form of acylcarnitines into the mitochondrial matrix and exit of free carnitine. The transport function of CACT is crucial for the β-oxidation pathway. In this work, it has been found that CACT is partially acetylated in rat liver mitochondria as demonstrated by anti-acetyl-lys antibody immunostaining. Acetylation was reversed by the deacetylase Sirtuin 3 in the presence of NAD⁺. After treatment of the mitochondrial extract with the deacetylase, the CACT activity, assayed in proteoliposomes, increased. The half-saturation constant of the CACT was not influenced, while the V _max was increased by deacetylation. Sirtuin 3 was not able to deacetylate the CACT when incubation was performed in intact mitoplasts, indicating that the acetylation sites are located in the mitochondrial matrix. Prediction on the localization of acetylated residues by bioinformatics correlates well with the experimental data. Recombinant CACT treated with acetyl-CoA was partially acetylated by non-enzymatic mechanism with a corresponding decrease of transport activity. The experimental data indicate that acetylation of CACT inhibits its transport activity, and thus may contribute to the regulation of the mitochondrial β-oxidation pathway.     

Interaction of beta-lactam antibiotics with the mitochondrial carnitine/acylcarnitine transporter

The interaction of beta-lactams with the purified mitochondrial carnitine/acylcarnitine transporter reconstituted in liposomes has been studied. Cefonicid, cefazolin, cephalothin, ampicillin, piperacillin externally added to the proteoliposomes, inhibited the carnitine/carnitine antiport catalysed by the reconstituted transporter. The most effective inhibitors were cefonicid and ampicillin with IC50 of 6.8 and 7.6mM, respectively. The other inhibitors exhibited IC50 values above 36 mM. Kinetic analysis performed with cefonicid and ampicillin revealed that the inhibition is completely competitive, i.e., the inhibitors interact with the substrate binding site. The Ki of the transporter is 4.9 mM for cefonicid and 9.9 mM for ampicillin. Cefonicid inhibited the transporter also on its internal side. The IC50 was 12.9 mM indicating that the inhibition was less pronounced than on the external side. Ampicillin and the other inhibitors were much less effective on the internal side. The beta-lactams were not transported by the carnitine/acylcarnitine transporter. Cephalosporins, and at much lower extent penicillins, caused irreversible inhibition of the transporter after prolonged time of incubation. The most effective among the tested antibiotics was cefonicid with IC50 of 0.12 mM after 60 h of incubation. The possible in vivo implications of the interaction of the beta-lactam antibiotics with the transporter are discussed.     

Analysis of essential histidine residues of maize branching enzymes by chemical modification and site-directed mutagenesis

Incubation of maize branching enzyme, mBEI and mBEII, with 100 μM diethylpyrocarbonate (DEPC) rapidly inactivated the enzymes. Treatment of the DEPC-inactivated enzymes with 100–500 mM hydroxylamine restored the enzyme activities. Spectroscopic data indicated that the inactivation of BE with DEPC was the result of histidine modification. The addition of the substrate amylose or amylopectin retarded the enzyme inactivation by DEPC, suggesting that the histidine residues are important for substrate binding. In maize BEII, conserved histidine residues are in catalytic regions 1 (His320) and 4 (His508). His320 and His508 were individually replaced by Ala via site-directed mutagenesis to probe their role in catalysis. Expression of these mutants inE. coli showed a significant decrease of the activity and the mutant enzymes hadK _m values 10 times higher than the wild type. Therefore, residues His320 and His508 do play an important role in substrate binding.     

Analysis of essential histidine residues of maize branching enzymes by chemical modification and site-directed mutagenesis

Incubation of maize branching enzyme, mBEI and mBEII, with 100 μM diethylpyrocarbonate (DEPC) rapidly inactivated the enzymes. Treatment of the DEPC-inactivated enzymes with 100–500 mM hydroxylamine restored the enzyme activities. Spectroscopic data indicated that the inactivation of BE with DEPC was the result of histidine modification. The addition of the substrate amylose or amylopectin retarded the enzyme inactivation by DEPC, suggesting that the histidine residues are important for substrate binding. In maize BEII, conserved histidine residues are in catalytic regions 1 (His320) and 4 (His508). His320 and His508 were individually replaced by Ala via site-directed mutagenesis to probe their role in catalysis. Expression of these mutants inE. coli showed a significant decrease of the activity and the mutant enzymes hadK _m values 10 times higher than the wild type. Therefore, residues His320 and His508 do play an important role in substrate binding.     

Glutathione controls the redox state of the mitochondrial carnitine/acylcarnitine carrier Cys residues by glutathionylation

Background

The mitochondrial carnitine/acylcarnitine carrier (CAC) is essential for cell metabolism since it catalyzes the transport of acylcarnitines into mitochondria allowing the β-oxidation of fatty acids. CAC functional and structural properties have been characterized. Cys residues which could form disulfides suggest the involvement of CAC in redox switches.

Methods

The effect of GSH and GSSG on the [³H]-carnitine/carnitine antiport catalyzed by the CAC in proteoliposomes has been studied. The Cys residues involved in the redox switch have been identified by site-directed mutagenesis. Glutathionylated CAC has been assessed by glutathionyl-protein specific antibody.

Results

GSH led to increase of transport activity of the CAC extracted from liver mitochondria. A similar effect was observed on the recombinant CAC. The presence of glutaredoxin-1 (Grx1) accelerated the GSH activation of the recombinant CAC. The effect was more evident at 37 °C. GSSG led to transport inhibition which was reversed by dithioerythritol (DTE). The effects of GSH and GSSG were studied on CAC Cys-mutants. CAC lacking C136 and C155 was insensitive to both reagents. Mutants containing these two Cys responded as the wild-type. Anti-glutathionyl antibody revealed the formation of glutathionylated CAC.

Conclusions

CAC is redox-sensitive and it is regulated by the GSH/GSSG couple. C136 and C155 are responsible for the regulation which occurs through glutathionylation.

General significance

CAC is sensitive to the redox state of the cell switching between oxidized and reduced forms in response to variation of GSSG and GSH concentrations.     

Solubilization and reconstitution of rat liver mitochondrial carnitine acylcarnitine translocase

Carnitine acylcarnitine translocase has been solubilized from inverted inner membrane vesicles of rat liver mitochondria with octyl glucoside and reconstituted into asolectin liposomes. For both processes, optimization of the detergent to phospholipid ratio was found crucial for obtaining reconstitutively active liposomes. Reassembly of the solubilized carrier into asolectin liposomes was achieved either by the octyl glucoside dilution method or by Extracti-Gel D column chromatography. The reconstituted system catalyzed exchange diffusion of carnitine, exhibited the expected inhibitor and temperature sensitivity, and discriminated between stereoisomers of octanoylcarnitine. The activity of unidirectional import of carnitine was low compared to exchange diffusion. It showed high-temperature sensitivity and a loss of activity on prolonged sonication that was regained by an appropriate freeze-thaw step subsequently.     

Site-directed mutagenesis of the His residues of the rat mitochondrial carnitine/acylcarnitine carrier: Implications for the role of His-29 in the transport pathway

The mitochondrial carnitine/acylcarnitine carrier (CAC) of Rattus norvegicus contains two His, His-29 and His-205. Only the first residue is conserved in all the members of the CAC subfamily and is positioned before the first of the three conserved motifs. In the homology model of CAC, His-29 is located in H1 close to the bottom of the central cavity. His-205 is the first amino acid of H5 and it is exposed towards the cytosol. The effect of substitution of the His residues on the transport function of the reconstituted mutant CACs has been analysed, in comparison with the wild-type. H29A showed very low activity, H29K and H29D were nearly inactive, whereas H205A, H205K and H205D showed activities similar to that of the wild-type. His-29 has also been substituted with Gln, Asn, Phe and Tyr. All the mutants showed very low transport function and, similarly to H29A, higher Km, reduced Vmax and altered selectivity towards (n)acylcarnitines, with the exception of H29Q, which exhibited functional properties similar to those of the wild-type. The experimental data, together with a comparative analysis of the carnitine acyltranferase active sites, indicated that His-29 forms an H-bond with the β-OH of carnitine. The substitution of His-205 led to a change of response of the CAC to the pH. The results are discussed in terms of relationships of His-29 with the molecular mechanism of translocation of the CAC.     

Role of cysteine residues in glutathione synthetase from Escherichia coli B. Chemical modification and oligonucleotide site-directed mutagenesis

Escherichia coli B glutathione synthetase is composed of four identical subunits; each subunit contains 4 cysteine residues (Cys-122, -195, -222, and -289). We constructed seven different mutant enzymes containing 3, 2, or no cysteine residues/subunit by replacement of cysteine codons with those of alanine in the gsh II gene using site-directed mutagenesis. Three mutant enzymes, Ala289, Ala222/289, Cys-free (Ala122/195/222/289), in which cysteine at residue 289 was replaced with alanine, were not inactivated by 5,5'-dithiobis(2-nitrobenzoate) (DTNB), while the other four mutants retaining Cys-289 were inactivated at the wild-type rate. From these selective inactivations of mutant enzymes by DTNB, the sulfhydryl group modified by DTNB was unambiguously identified as Cys-289. In this way, Cys-289 was found to be also a target of modification with 2-nitrothiocyanobenzoate and N-ethylmaleimide, while Cys-195 was of p-chloromercuribenzoate. These results suggest that both Cys-195 and Cys-289 were not essential for the activity of the glutathione synthetase, but chemical modification of either one of the two sulfhydryl groups resulted in complete loss of the activity. Replacement of Cys-122 to Ala-122 enhanced the reactivity of Cys-289 with sulfhydryl reagents.     

Identification of essential histidine residues in 3-deoxy-D-manno-octulosonic acid 8-phosphate synthase: analysis by chemical modification with diethyl pyrocarbonate and site-directed mutagenesis

The enzyme 3-deoxy-D-manno-octulosonic acid 8-phosphate (KDO 8-P) synthase from Escherichia coli that catalyzes the aldol-type condensation of D-arabinose 5-phosphate (A 5-P) and phosphoenolpyruvate (PEP) to give KDO 8-P and inorganic phosphate (P(i)) is inactivated by diethyl pyrocarbonate (DEPC). The inactivation is first-order in enzyme and DEPC. A second-order rate constant of 340 M(-1) min(-1) is obtained at pH 7.6 and 4 degrees C. The rate of inactivation is dependent on pH and the pH-inactivation rate data imply the involvement of an amino acid residue with a pK(a) value of 7.3. KDO 8-P synthase activity is not restored to the DEPC-inactivated enzyme following treatment with hydroxylamine. Complete loss of KDO 8-P synthase activity correlates with the ethoxyformylation of three histidine residues by DEPC. KDO 8-P synthase is protected against DEPC inactivation by PEP and partially protected against inactivation by A 5-P. To provide further evidence for the involvement or role of the histidine residues in the aldol-type condensation catalyzed by KDO 8-P synthase, all six histidines were individually mutated to either glycine or alanine. The kinetic constants for the three mutants H40A, H67G, and H246G were unaffected as compared to the wild type enzyme. In contrast, H241G demonstrates a >10-fold increase in K(M) for both PEP and A 5-P and a 4-fold reduction in k(cat), while H97G demonstrates an increase in K(M) for only A 5-P and a 2-fold reduction in k(cat). The activity of the H202G mutant was too low to be measured accurately but the data obtained indicated an approximate 400-fold reduction in k(cat). Circular dichroism measurements of the wild-type and mutant enzymes indicate modest structural changes in only the fully active H67G and H246G mutants. The H241G mutant is protected against DEPC inactivation by PEP and A 5-P to the same extent as the wild-type enzyme, suggesting that the functionally important H241 may not be located in the vicinity of the substrate binding sites. The H97G mutant is protected by PEP against DEPC inactivation to the same degree as the wild-type enzyme but is no longer protected by A 5-P. In the case of the H202G mutant, both A 5-P and PEP protect the mutant against DEPC inactivation but to different extents from those observed for the wild-type enzyme. The catalytic activity of the H97G mutant is partially restored (20% --> 60% of wild-type activity) in the presence of imidazole, while a minor amount of activity is restored to the H202G mutant (<1% --> 4% of wild-type activity) in the presence of imidazole.     

Evaluation of the role of electrostatic residues in human epidermal growth factor by site-directed mutagenesis and chemical modification.

Four residues in the carboxy-terminal domain of human epidermal growth factor (hEGF), glutamate 40, glutamine 43, arginine 45, and aspartate 46 were targeted for site-directed mutagenesis to evaluate their potential role in epidermal growth factor (EGF) receptor-ligand interaction. One or more mutations were generated at each of these sites and the altered recombinant hEGF gene products were purified and evaluated by radioreceptor competition binding assay. Charge-conservative replacement of glutamate 40 with aspartate resulted in a decrease in receptor binding affinity to 30% relative to wild-type hEGF. On the other hand, removal of the electrostatic charge by substitution of glutamate 40 with glutamine or alanine resulted in only a slightly greater decrease in receptor binding to 25% relative receptor affinity. The introduction of a positive charge upon substitution of glutamine 43 with lysine had no effect on receptor binding. The substitution of arginine 45 with lysine also showed no effect on receptor binding, unlike the absolute requirement observed for the arginine side-chain at position 41 [Engler DA, Campion SR, Hauser MR, Cook JS, Niyogi, SK: J Biol Chem 267:2274-2281, 1992]. Subsequent elimination of the positive charge of lysine 45 by reaction with potassium cyanate showed that the electrostatic property of the residue at this site, as well as that at lysine 28 and lysine 48, was not required for receptor-ligand association.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of histidine residues in EcoP15I DNA methyltransferase activity as probed by chemical modification and site-directed mutagenesis

Towards understanding the catalytic mechanism of M.EcoP15I [EcoP15I MTase (DNA methyltransferase); an adenine methyltransferase], we investigated the role of histidine residues in catalysis. M.EcoP15I, when incubated with DEPC (diethyl pyrocarbonate), a histidine-specific reagent, shows a time- and concentration-dependent inactivation of methylation of DNA containing its recognition sequence of 5'-CAGCAG-3'. The loss of enzyme activity was accompanied by an increase in absorbance at 240 nm. A difference spectrum of modified versus native enzyme shows the formation of N-carbethoxyhistidine that is diminished by hydroxylamine. This, along with other experiments, strongly suggests that the inactivation of the enzyme by DEPC was specific for histidine residues. Substrate protection experiments show that pre-incubating the methylase with DNA was able to protect the enzyme from DEPC inactivation. Site-directed mutagenesis experiments in which the 15 histidine residues in the enzyme were replaced individually with alanine corroborated the chemical modification studies and established the importance of His-335 in the methylase activity. No gross structural differences were detected between the native and H335A mutant MTases, as evident from CD spectra, native PAGE pattern or on gel filtration chromatography. Replacement of histidine with alanine residue at position 335 results in a mutant enzyme that is catalytically inactive and binds to DNA more tightly than the wild-type enzyme. Thus we have shown in the present study, through a combination of chemical modification and site-directed mutagenesis experiments, that His-335 plays an essential role in DNA methylation catalysed by M.EcoP15I.     

Identification of residues involved in v-Src substrate recognition by site-directed mutagenesis.

To study the role of the catalytic domain in v-Src substrate specificity, we engineered three site-directed mutants (Leu-472 to Tyr or Trp and Thr-429 to Met). The mutant forms of Src were expressed in Sf9 cells and purified. We analyzed the substrate specificities of wild-type v-Src and the mutants using two series of peptides that varied at residues C-terminal to tyrosine. The peptides contained either the YMTM motif found in insulin receptor substrate-1 (IRS-1) or the YGEF motif identified from peptide library experiments to be the optimal sequence for Src. Mutations at positions Leu-472 or Thr-429 caused changes in substrate specificity at positions P+1 and P+3 (i.e. one or three residues C-terminal to tyrosine). This was particularly evident in the case of the L-472W mutant, which had pronounced alterations in its preferences at the P+1 position. The results suggest that residue Leu-472 plays a role in P+1 substrate recognition by Src. We discuss the results in the light of recent work on the roles of the SH2, SH3 and catalytic domains of Src in substrate specificity.     

Identification of two cysteine residues forming a pair of vicinal thiols in glucosamine-6-phosphate deaminase from Escherichia coli and a study of their functional role by site-directed mutagenesis.

The nucleotide sequence of the nagB gene in Escherichia coli, encoding glucosamine-6-phosphate deaminase, located four cysteinyl residues at positions 118, 219, 228, and 239. Chemical modification studies performed with the purified enzyme had shown that the sulfhydryl groups of two of these residues form a vicinal pair in the enzyme and are easily modified by thiol reagents. The allosteric transition to the more active conformer (R), produced by the binding of homotropic (D-glucosamine 6-phosphate or 2-deoxy-2-amino-D-glucitol 6-phosphate) or heterotropic (N-acetyl-D-glucosamine 6-phosphate) ligands, completely protected these thiols against chemical modification. Selective cyanylation of the vicinal thiols with 2-nitro-5-(thiocyanato)benzoate, followed by alkaline hydrolysis to produce chain cleavage at the modified cysteines, gave a pattern of polypeptides which allowed us to identify Cys118 and Cys239 as the residues forming the thiol pair. Subsequently, three mutated forms of the gene were constructed by oligonucleotide-directed mutagenesis, in which one or both of the cysteine codons were changed to serine. The mutant proteins were overexpressed and purified, and their kinetics were studied. The dithiol formed by Cys118 and Cys239 was necessary for maximum catalytic activity. The single replacements and the double mutation affected catalytic efficiency in a similar way, which was also identical to the effect of the chemical block of the thiol pair. However, only one of these cysteinyl residues, Cys239, had a significant role in the allosteric transition, and its substitution for serine reduced the allosteric interaction energy, due to a lower value of KT.     

Role of cysteine residues in ribonuclease H from Escherichia coli. Site-directed mutagenesis and chemical modification.

The role of the three cysteine residues at positions 13, 63 and 133 in Escherichia coli RNAase H, an enzyme that is sensitive to N-ethylmaleimide [Berkower, Leis & Hurwitz (1973) J. Biol. Chem. 248, 5914-5921], was examined by using both site-directed mutagenesis and chemical modification. Novel aspects that were found are as follows. First, none of the cysteine residues is required for activity. Secondly, chemical modification of either Cys-13 or Cys-133 with thiol-blocking reagents inactivates the enzyme, but that of Cys-63 does not. Thus the sensitivity of E. coli RNAase H to N-ethylmaleimide arises not from blocking of the thiol group but from steric hindrance by the modifying group incorporated at either Cys-13 or Cys-133.     

Interaction of mildronate with the mitochondrial carnitine/acylcarnitine transport protein

The interaction of mildronate [3-(2,2,2-trimethylhydrazine) propionate] with the purified mitochondrial carnitine/acylcarnitine transporter reconstituted in liposomes has been studied. Mildronate, externally added to the proteoliposomes, strongly inhibited the carnitine/carnitine antiport catalyzed by the reconstituted transporter with an IC(50) of 560 muM. A kinetic analysis revealed that the inhibition is completely competitive, that is, mildronate interacts with the substrate-binding site. The half-saturation constant of the transporter for external mildronate (K(i)) is 530 muM. Carnitine/mildronate antiport has been measured as [(3)H]carnitine uptake into proteoliposomes containing internal mildronate or as [(3)H]carnitine efflux from proteoliposomes in the presence of external mildronate, indicating that mildronate is transported by the carnitine/acylcarnitine transporter and that the inhibition observed was due to the transport of mildronate in the place of carnitine. The intraliposomal half-saturation constant for mildronate transport (K(m)) has been determined. Its value, 18 mM, is much higher than the external half-saturation constant (K(i)) in agreement with the asymmetric properties of the transporter. In vivo, the antiport reaction between cytosolic (administered) mildronate and matrix carnitine may cause intramitochondrial carnitine depletion. This effect, together with the inhibition of the physiological transport, will lead to impairment of fatty acid utilization.     

Stabilization of NAD-dependent formate dehydrogenase from Candida boidinii by site-directed mutagenesis of cysteine residues.

The gene of the NAD-dependent formate dehydrogenase (FDH) from the yeast Candida boidinii was cloned by PCR using genomic DNA as a template. Expression of the gene in Escherichia coli yielded functional FDH with about 20% of the soluble cell protein. To confirm the hypothesis of a thiol-coupled inactivation process, both cysteine residues in the primary structure of the enzyme have been exchanged by site-directed mutagenesis using a homology model based on the 3D structure of FDH from Pseudomonas sp. 101 and from related dehydrogenases. Compared to the wt enzyme, most of the mutants were significantly more stable towards oxidative stress in the presence of Cu(II) ions, whereas the temperature optima and kinetic constants of the enzymatic reaction are not significantly altered by the mutations. Determination of the Tm values revealed that the stability at temperatures above 50 degrees C is optimal for the native and the recombinant wt enzyme (Tm 57 degrees C), whereas the Tm values of the mutant enzymes vary in the range 44-52 degrees C. Best results in initial tests concerning the application of the enzyme for regeneration of NADH in biotransformation of trimethyl pyruvate to Ltert leucine were obtained with two mutants, FDHC23S and FDHC23S/C262A, which are significantly more stable than the wt enzyme.     

Identification of residues essential for progesterone binding to uteroglobin by site-directed mutagenesis

In order to identify amino acids directly involved in progesterone binding to rabbit uteroglobin we have mutated Phe 6, Tyr 21 and Thr 60 by site-directed mutagenesis of the uteroglobin cDNA. These residues have been postulated previously to participate in progesterone binding. High-level expression of the mutated uteroglobin cDNAs in Escherichia coli yields recombinant protein mutants that, like natural uteroglobin, form stable dimers, suggesting that the tertiary structure of the protein has not been altered. Substitution of Phe 6 by Ser or Ala does not change the progesterone binding characteristics. In contrast, replacement of Tyr 21 by Phe or Ala, drastically decreases progesterone binding. In addition, replacement of Thr 60 by Ala reduces the affinity for progesterone by a factor of three. These data suggest a direct interaction of progesterone with these two amino acids and support the idea of direct hydrogen bonding of the carbonyl (C3 and C20) of progesterone with the hydroxyl groups of Tyr 21 and Thr 60, respectively.     

Identification of catalytically essential residues in Escherichia coli esterase by site-directed mutagenesis.

Escherichia coli esterase (EcE) is a member of the hormone-sensitive lipase family. We have analyzed the roles of the conserved residues in this enzyme (His103, Glu128, Gly163, Asp164, Ser165, Gly167, Asp262, Asp266 and His292) by site-directed mutagenesis. Among them, Gly163, Asp164, Ser165, and Gly167 are the components of a G-D/E-S-A-G motif. We showed that Ser165, Asp262, and His292 are the active-site residues of the enzyme. We also showed that none of the other residues, except for Asp164, is critical for the enzymatic activity. The mutation of Asp164 to Ala dramatically reduced the catalytic efficiency of the enzyme by the factor of 10(4) without seriously affecting the substrate binding. This residue is probably structurally important to make the conformation of the active-site functional.