Sites of pH regulation of the urea channel of Helicobacter pylori

Helicobacter pylori (Hp) and Streptococcus salivarius (Ss) require intrabacterial urease for acid resistance and express a urea channel, UreI. The presence of UreI was shown to increase urea permeability approximately 300-fold over that of a non-polar ureI deletion mutant. Expression of SsUreI in Xenopus oocytes increased urea uptake pH independently, whereas HpUreI shows an acidic pH dependence, half-maximal at pH 6.0. Mutagenesis of all histidines, aspartates, glutamates and the lysine in the periplasmic domain of HpUreI showed that His-123, His-131, Asp-129, Asp-140, Glu-138 and Lys-132 in the second periplasmic loop (PL2) and His-193 in the C-terminus (Ct) were important for activation of transport. With the exception of a lysine that was shown to substitute for His-193 in HpUreI, these charged amino acids are absent in SsUreI. A chimera in which PL1 of HpUreI was replaced by PL1 of SsUreI retained activity at acidic pH and gained partial activity at neutral pH. Exchange of PL2 inactivated transport, whereas exchange of Ct had no effect. Chimeras, in which either PL1 or PL2 of HpUreI replaced those of SsUreI, retained wild-type transport, but replacement of the Ct or both loops inactivated transport. PL1 appears to be important for restricting transport through HpUreI at neutral pH, whereas protonation of three histidines in PL2 and Ct and the presence of three dicarboxylic amino acids in PL2 appears to be necessary to activate HpUreI at acidic pH.     

Mutational analysis of the MutH protein from Escherichia coli

Site-directed mutagenesis was performed on several areas of MutH based on the similarity of MutH and PvuII structural models. The aims were to identify DNA-binding residues; to determine whether MutH has the same mechanism for DNA binding and catalysis as PvuII; and to localize the residues responsible for MutH stimulation by MutL. No DNA-binding residues were identified in the two flexible loop regions of MutH, although similar loops in PvuII are involved in DNA binding. Two histidines in MutH are in a similar position as two histidines (His-84 and His-85) in PvuII that signal for DNA binding and catalysis. These MutH histidines (His-112 and His-115) were changed to alanines, but the mutant proteins had wild-type activity both in vivo and in vitro. The results indicate that the MutH signal for DNA binding and catalysis remains unknown. Instead, a lysine residue (Lys-48) was found in the first flexible loop that functions in catalysis together with the three presumed catalytic amino acids (Asp-70, Glu-77, and Lys-79). Two deletion mutations (MutHDelta224 and MutHDelta214) in the C-terminal end of the protein, localized the MutL stimulation region to five amino acids (Ala-220, Leu-221, Leu-222, Ala-223, and Arg-224).     

Crystal structural analysis and metal-dependent stability and activity studies of the ColE7 endonuclease domain in complex with DNA/Zn2+ or inhibitor/Ni2+

The nuclease domain of ColE7 (N-ColE7) contains an H-N-H motif that folds in a beta beta alpha-metal topology. Here we report the crystal structures of a Zn2+-bound N-ColE7 (H545E mutant) in complex with a 12-bp duplex DNA and a Ni2+-bound N-ColE7 in complex with the inhibitor Im7 at a resolution of 2.5 A and 2.0 A, respectively. Metal-dependent cleavage assays showed that N-ColE7 cleaves double-stranded DNA with a single metal ion cofactor, Ni2+, Mg2+, Mn2+, and Zn2+. ColE7 purified from Escherichia coli contains an endogenous zinc ion that was not replaced by Mg2+ at concentrations of <25 mM, indicating that zinc is the physiologically relevant metal ion in N-ColE7 in host E. coli. In the crystal structure of N-ColE7/DNA complex, the zinc ion is directly coordinated to three histidines and the DNA scissile phosphate in a tetrahedral geometry. In contrast, Ni2+ is bound in N-ColE7 in two different modes, to four ligands (three histidines and one phosphate ion), or to five ligands with an additional water molecule. These data suggest that the divalent metal ion in the His-metal finger motif can be coordinated to six ligands, such as Mg2+ in I-PpoI, Serratia nuclease and Vvn, five ligands or four ligands, such as Ni2+ or Zn2+ in ColE7. Universally, the metal ion in the His-metal finger motif is bound to the DNA scissile phosphate and serves three roles during hydrolysis: polarization of the P-O bond for nucleophilic attack, stabilization of the phosphoanion transition state and stabilization of the cleaved product.     

Conserved residues in the putative catalytic triad of human bile acid Coenzyme A:amino acid N-acyltransferase

Human bile acid-CoA:amino acid N-acyltransferase (hBAT), an enzyme catalyzing the conjugation of bile acids with the amino acids glycine or taurine has significant sequence homology with dienelactone hydrolases and other alpha/beta hydrolases. These enzymes have a conserved catalytic triad that maps onto the mammalian BATs at residues Cys-235, Asp-328, and His-362 of the human sequence, albeit that the hydrolases contain a serine instead of a cysteine. In the present study, the function of the putative catalytic triad of hBAT was examined by chemical modification with the cysteine alkylating reagent N-ethylmaleimide (NEM) and by site-directed mutagenesis of the triad residues followed by enzymology studies of mutant and wild-type hBATs. Treatment with NEM caused inactivation of wild-type hBAT. However, preincubation of wild-type hBAT with the substrate cholyl-CoA before NEM treatment prevented loss of N-acyltransferase activity. Substitution of His-362 or Asp-328 with alanine results in inactivation of hBAT. Although substitution of Cys-235 with serine generated an hBAT mutant with lower N-acyltransferase activity, it substantially increased the bile acid-CoA thioesterase activity compared with wild type. In summary, data from this study support the existence of an essential catalytic triad within hBAT consisting of Cys-235, His-362, and Asp-328 with Cys-235 serving as the probable nucleophile and thus the site of covalent attachment of the bile acid molecule.     

Site-specific mutations of conserved C-terminal residues in aminoglycoside 3'-phosphotransferase II: phenotypic and structural analysis of mutant enzymes.

In order to test the biological importance of amino acids in the C-terminal quarter of aminoglycoside 3'-phosphotransferase II enzyme, seven of the highly conserved residues in this region, His-188, Asp-190, Asp-208, Gly-210, Arg-211, Asp-216 and Asp-220, were changed via site-directed mutagenesis. The phenotype of each mutant was compared to wildtype in terms of antibiotic susceptibilities and enzymatic activities. All of the substitutions either abolished or significantly reduced the resistance of the resulting strains to kanamycin, neomycin, butirosin, ribostamycin, paromomycin, gentamicin A, and G-418. Similarly, enzyme activities in crude extracts were substantially reduced for the mutant strains. Affinity of the enzyme for Mg+2-ATP decreased with His-188, Asp-190, Asp-216 and Asp-220 substitutions as revealed by Km measurements. Secondary structure analysis predicted that substitutions at the conserved residues caused severe conformational distortions at the corresponding regions of the protein.     

beta-Ketoacyl-[acyl carrier protein] synthase I of Escherichia coli: aspects of the condensation mechanism revealed by analyses of mutations in the active site pocket

beta-Ketoacyl-[acyl carrier protein (ACP)] synthase forms new carbon-carbon bonds in three steps: transfer of an acyl primer from ACP to the enzyme, decarboxylation of the elongating substrate and its condensation with the acyl primer substrate. Six residues of Escherichia coli beta-ketoacyl-ACP synthase I (KAS I) implicated in these reactions were subjected to site-directed mutagenesis. Analyses of the abilities of C163A, C163S, H298A, D306A, E309A, K328A, and H333A to carry out the three reactions lead to the following conclusions. The active site Cys-163 is not required for decarboxylation, whereas His-298 and His-333 are indispensable. Neither of the histidines is essential for increasing the nucleophilicity of Cys-163 to enable transfer of the acyl primer substrate. Maintenance of the structural integrity of the active site by Asp-306 and Glu-309 is required for decarboxylation but not for transfer. One function of Lys-328 occurs very early in catalysis, potentially before transfer. These results in conjunction with structural analyses of substrate complexes have led to a model for KAS I catalysis [Olsen, J. G., Kadziola, A., von Wettstein-Knowles, P., Siggaard-Andersen, M., and Larsen, S. (2001) Structure 9, 233-243]. Another facet of catalysis revealed by the mutant analyses is that the acyl primer transfer activity of beta-ketoacyl-ACP synthase I is inhibited by free ACP at physiological concentrations. Differences in the inhibitory response by individual mutant proteins indicate that interaction of free ACP with Cys-163, Asp-306, Glu-309, Lys-328, and His-333 might form a sensitive regulatory mechanism for the transfer of acyl primers.     

Mutation analysis of the histidine residues in the glycylglycine endopeptidase ALE-1

A novel staphylolytic enzyme, ALE-1, is a glycylglycine endopeptidase produced by Staphylococcus capitis EPK1. ALE-1 possesses seven histidines. Chemical modification studies using diethylpyrocarbonate and iodoacetic acid suggested that a histidine or tyrosine residue(s) in the molecule is important for the organism's staphylolytic activity. All of the histidine residues, one tyrosine, and one aspartic acid residue in the N-terminally truncated ALE-1 (DeltaN-term ALE-1) were systematically altered by site-directed mutagenesis, and the enzyme activities and metal contents of the variants were measured. Our studies indicated that His-150, His-200, His-231, His-233, and Asp-154 are essential for the enzyme activity of DeltaN-term ALE-1. Except for His-150 and Asp-154, all of these amino acids were located within the 38-amino-acid region conserved among 11 proteins, including 5 staphylolytic endopeptidases. Inductively coupled plasma-mass spectrometric analysis of DeltaN-term ALE-1 revealed that it contains one atom of zinc per molecule. Measurement of the zinc content of the mutant DeltaN-term ALE-1 suggested that His-150 and -233 are important for zinc binding; their loss in these variant enzymes coincided with the loss of staphylolytic activity. These results strongly suggest that ALE-1 is a novel member of zinc metalloproteases.     

Molecular switches for pheromone release from a moth pheromone-binding protein

Pheromone-binding proteins (PBPs) are involved in the uptake of pheromones from pores on the antennae, transport through an aqueous environment surrounding the olfactory receptor neurons, and fast delivery to pheromone receptors. We tested the hypothesis that a C-terminal segment and a flexible loop are involved in the release of pheromones to membrane-bound receptors. We expressed in Escherichia coli 11 mutants of the PBP from the silkworm moth, BmorPBP, taking into consideration structural differences between the forms with high and low binding affinity. The N-terminus was truncated and His-69, His-70 and His-95 at the base of a flexible loop, and a cluster of acidic residues at the C-terminus were mutated. Binding assays and circular dichroism analyses support a mechanism involving protonation of acidic residues Asp-132 and Glu-141 at the C-terminus and histidines, His-70 and His-95, in the base of a loop covering the binding pocket. The former leads to the formation of a new α-helix, which competes with pheromone for the binding pocket, whereas positive charge repulsion of the histidines opens the opposite side of the binding pocket.     

Cysteine-scanning mutagenesis of muscle carnitine palmitoyltransferase I reveals a single cysteine residue (Cys-305) is important for catalysis

Carnitine palmitoyltransferase (CPT) I catalyzes the conversion of long-chain fatty acyl-CoAs to acyl carnitines in the presence of l-carnitine, a rate-limiting step in the transport of long-chain fatty acids from the cytoplasm to the mitochondrial matrix. To determine the role of the 15 cysteine residues in the heart/skeletal muscle isoform of CPTI (M-CPTI) on catalytic activity and malonyl-CoA sensitivity, we constructed a 6-residue N-terminal, a 9-residue C-terminal, and a 15-residue cysteineless M-CPTI by cysteine-scanning mutagenesis. Both the 9-residue C-terminal mutant enzyme and the complete 15-residue cysteineless mutant enzyme are inactive but that the 6-residue N-terminal cysteineless mutant enzyme had activity and malonyl-CoA sensitivity similar to those of wild-type M-CPTI. Mutation of each of the 9 C-terminal cysteines to alanine or serine identified a single residue, Cys-305, to be important for catalysis. Substitution of Cys-305 with Ala in the wild-type enzyme inactivated M-CPTI, and a single change of Ala-305 to Cys in the 9-residue C-terminal cysteineless mutant resulted in an 8-residue C-terminal cysteineless mutant enzyme that had activity and malonyl-CoA sensitivity similar to those of the wild type, suggesting that Cys-305 is the residue involved in catalysis. Sequence alignments of CPTI with the acyltransferase family of enzymes in the GenBank led to the identification of a putative catalytic triad in CPTI consisting of residues Cys-305, Asp-454, and His-473. Based on the mutagenesis and substrate labeling studies, we propose a mechanism for the acyltransferase activity of CPTI that uses a catalytic triad composed of Cys-305, His-473, and Asp-454 with Cys-305 serving as a probable nucleophile, thus acting as a site for covalent attachment of the acyl molecule and formation of a stable acyl-enzyme intermediate. This would in turn allow carnitine to act as a second nucleophile and complete the acyl transfer reaction.     

Unique disulfide bonds in epidermal growth factor (EGF) domains of β3 affect structure and function of αIIbβ3 and αvβ3 integrins in different manner

The β3 subunit of αIIbβ3 and αvβ3 integrins contains four epidermal growth factor (EGF)-like domains. Each domain harbors four disulfide bonds of which one is unique for integrins. We previously discerned a regulatory role of the EGF-4 Cys-560-Cys-583 unique bond for αIIbβ3 activation. In this study we further investigated the role of all four integrin unique bonds in both αIIbβ3 and αvβ3. We created β3 mutants harboring serine substitutions of each or both cysteines that disrupt the four unique bonds (Cys-437-Cys-457 in EGF-1, Cys-473-Cys-503 in EGF-2, Cys-523-Cys-544 in EGF-3, and Cys-560-Cys-583 in EGF-4) and transfected them into baby hamster kidney cells together with normal αv or αIIb. Flow cytometry was used to measure surface expression of αIIbβ3 and αvβ3 and their activity state by soluble fibrinogen binding. Most cysteine substitutions caused similarly reduced surface expression of both receptors. Disrupting all four unique disulfide bonds by single cysteine substitutions resulted in variable constitutive activation of αIIbβ3 and αvβ3. In contrast, whereas double C437S/C457S and C473S/C503S mutations yielded constitutively active αIIbβ3 and αvβ3, the C560S/C583S mutation did not, and the C523S/C544S mutation only yielded constitutively active αIIbβ3. Activation of C523S/C544S αvβ3 mutant by activating antibody and dithiothreitol was also impaired. Molecular dynamics of C523S/C544S β3 in αIIbβ3 but not in αvβ3 displayed an altered stable conformation. Our findings indicate that unique disulfide bonds in β3 differently affect the function of αIIbβ3 and αvβ3 and suggest a free sulfhydryl-dependent regulatory role for Cys-560-Cys-583 in both αIIbβ3 and αvβ3 and for Cys-523-Cys-544 only in αvβ3.     

Site-directed mutagenesis of the putative catalytic triad of poliovirus 3C proteinase.

Based on predictions of the structure of proteinase 3C of poliovirus, mutations have been made at residues that are supposed to constitute the catalytic triad. Wild-type and mutant 3C were expressed in Escherichia coli, purified to homogeneity, and characterized by the ability to cleave a synthetic peptide substrate or an in vitro translated polypeptide consisting of part of the polyprotein of poliovirus. Additionally, the ability of autocatalytic processing of a precursor harboring wild-type or mutant 3C sequences was tested. Single substitutions of the residues His-40, Glu-71, and Cys-147 by Tyr, Gln, and Ser, respectively, resulted in an inactive enzyme. Replacement of Asp-85 by Asn resulted in an enzyme that was as active as wild-type enzyme in trans cleavage assays but whose autoprocessing ability was impaired. Our results are consistent with the proposal that residues His-40, Glu-71, and Cys-147 constitute the catalytic triad of poliovirus 3C proteinase. Furthermore, residue Asp-85 is not required for proper proteolytic activity despite being highly conserved between different picornaviruses. This indicates that Asp-85 might be involved in a different function of 3C.     

Crystal structure of D-aminoacylase from Alcaligenes faecalis DA1. A novel subset of amidohydrolases and insights into the enzyme mechanism

D-Aminoacylase is an attractive candidate for commercial production of D-amino acids through its catalysis in the hydrolysis of N-acyl-D-amino acids. We report here the first D-aminoacylase crystal structure from A. faecalis at 1.5-A resolution. The protein comprises a small beta-barrel, and a catalytic (betaalpha)(8)-barrel with a 63-residue insertion. The enzyme structure shares significant similarity to the alpha/beta-barrel amidohydrolase superfamily, in which the beta-strands in both barrels superimpose well. Unexpectedly, the enzyme binds two zinc ions with widely different affinities, although only the tightly bound zinc ion is required for activity. One zinc ion is coordinated by Cys(96), His(220), and His(250), while the other is loosely chelated by His(67), His(69), and Cys(96). This is the first example of the metal ion coordination by a cysteine residue in the superfamily. Therefore, D-aminoacylase defines a novel subset and is a mononuclear zinc metalloenzyme but containing a binuclear active site. The preferred substrate was modeled into a hydrophobic pocket, revealing the substrate specificity and enzyme catalysis. The 63-residue insertion containing substrate-interacting residues may act as a gate controlling access to the active site, revealing that the substrate binding would induce a closed conformation to sequester the catalysis from solvent.     

Site-directed mutagenesis of conserved amino acids in the alpha subunit of toluene dioxygenase: potential mononuclear non-heme iron coordination sites.

The terminal oxygenase component of toluene dioxygenase from Pseudomonas putida F1 is an iron-sulfur protein (ISP(TOL)) that requires mononuclear iron for enzyme activity. Alignment of all available predicted amino acid sequences for the large (alpha) subunits of terminal oxygenases showed a conserved cluster of potential mononuclear iron-binding residues. These were between amino acids 210 and 230 in the alpha subunit (TodC1) of ISP(TOL). The conserved amino acids, Glu-214, Asp-219, Tyr-221, His-222, and His-228, were each independently replaced with an alanine residue by site-directed mutagenesis. Tyr-266 in TodC1, which has been suggested as an iron ligand, was treated in an identical manner. To assay toluene dioxygenase activity in the presence of TodC1 and its mutant forms, conditions for the reconstitution of wild-type ISP(TOL) activity from TodC1 and purified TodC2 (beta subunit) were developed and optimized. A mutation at Glu-214, Asp-219, His-222, or His-228 completely abolished toluene dioxygenase activity. TodC1 with an alanine substitution at either Tyr-221 or Tyr-266 retained partial enzyme activity (42 and 12%, respectively). In experiments with [14C]toluene, the two Tyr-->Ala mutations caused a reduction in the amount of Cis-[14C]-toluene dihydrodiol formed, whereas a mutation at Glu-214, Asp-219, His-222, or His-228 eliminated cis-toluene dihydrodiol formation. The expression level of all of the mutated TWO proteins was equivalent to that of wild-type TodC1 as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot (immunoblot) analyses. These results, in conjunction with the predicted amino acid sequences of 22 oxygenase components, suggest that the conserved motif Glu-X3-4,-Asp-X2-His-X4-5-His is critical for catalytic function and the glutamate, aspartate, and histidine residues may act as mononuclear iron ligands at the site of oxygen activation.     

Structure of a ternary complex of proteinase K,mercury, and a substrate-analogue hexa-peptide at 2.2 Å resolution

The crystal structure of a ternary complex of proteinase K, Hg(II) and a hexapeptide N-Ac-Pro-Ala-Pro-Phe-Pro-Ala-NH₂ has been determined at 2.2 Å resolution and refined to an R factor of 0.172 for 12,910 reflections. The mercury atom occupies two alternate sites, each of which was assigned an occupancy of 0.45. These two sites are bridged by Cys-73 Sγ which forms covalent bonds to both. Both mercury sites form regular polyhedrons involving atoms from residues Asp-39, His-69, Cys-73, His-72, Met-225, and Wat-324. The complex formation with mercury seems to disturb the stereochemistry of the residues of the catalytic triad Asp-39, His-69, and Ser-224 appreciably, thus reducing the enzymatic activity of proteinase K to 15%. The electron density in the difference Fourier map shows that the hexapeptide occupies the S1 subsite predominantly and the standard recognition site constituted by Ser-132 to Gly-136 and Gly-100 to Tyr-104 segments is virtually empty. The hexapeptide is held firmly through a series of hydrogen bonds involving protein atoms and water molecules. As a result of complex formation, Asp-39, His-69, Met-225, Ile-220, Ser-219, Thr-223, and Ser-224 residues move appreciably to accommodate the mercury atoms and the hexapeptide. The largest movement is observed for Met-225 which is involved in multiple interactions with both mercury and the hexapeptide. The activity results indicate an inhibition rate of 95%, as a result of the combined effect of mercury and hexapeptide. © 1996 Wiley-Liss, Inc.     

Site-directed mutagenesis suggests close functional relationship between a human rhinovirus 3C cysteine protease and cellular trypsin-like serine proteases

Human rhinoviruses, like other picornaviruses, encode a cysteine protease (designated 3C) which cleaves mainly at viral Gln-Gly pairs. There are significant areas of homology between picornavirus 3C cysteine proteases and cellular serine proteases (e.g. trypsin), suggesting a functional relationship between their catalytic regions. To test this functional relationship, we made single substitutions in human rhinovirus type 14 protease 3C at seven amino acid positions which are highly conserved in the 3C proteases of animal picornaviruses. Substitutions at either His-40, Asp-85, or Cys-146, equivalent to the trypsin catalytic triad His-57, Asp-102, and Ser-195, respectively, completely abolished 3C proteolytic activity. Single substitutions were also made at either Thr-141, Gly-158, His-160, or Gly-162, which are equivalent to the trypsin specificity pocket region. Only the mutant with a conservative Thr-141 to Ser substitution exhibited proteolytic activity, which was much reduced compared with the parent. These results, together with immunoprecipitation data which indicate that Asp-85, Thr-141, and Cys-146 lie in accessible surface regions, suggest that the catalytic mechanism of picornavirus 3C cysteine proteases is closely related to that of cellular trypsin-like serine proteases.     

Role of aspartate-133 and histidine-458 in the mechanism of tryptophan indole-lyase from Proteus vulgaris

Tryptophan indole-lyase (Trpase) from Proteus vulgaris is a pyridoxal 5'-phosphate dependent enzyme that catalyzes the reversible hydrolytic cleavage of L-Trp to yield indole and ammonium pyruvate. Asp-133 and His-458 are strictly conserved in all sequences of Trpase, and they are located in the proposed substrate-binding region of Trpase. These residues were mutated to alanine to probe their role in substrate binding and catalysis. D133A mutant Trpase has no measurable activity with L-Trp as substrate, but still retains activity with S-(o-nitrophenyl)-L-cysteine, S-alkyl-L-cysteines, and beta-chloro-L-alanine. H458A mutant Trpase has 1.6% of wild-type Trpase activity with L-Trp, and high activity with S-(o-nitrophenyl)-L-cysteine, S-alkyl-L-cysteines, and beta-chloro-L-alanine. H458A mutant Trpase does not exhibit the pK(a) of 5.3 seen in the pH dependence of k(cat)/K(m) of L-Trp for wild-type Trpase. Both mutant enzymes are inhibited by L-Ala, L-Met, and L-Phe, with K(i) values similar to those of wild-type Trpase, but oxindolyl-L-alanine and beta-phenyl-DL-serine show much weaker binding to the mutant enzymes, suggesting that Asp-133 and His-458 are involved in the binding of these ligands. D133A and H458A mutant Trpase exhibit absorption and CD spectra in the presence of substrates and inhibitors that are similar to wild-type Trpase, with peaks at about 420 and 500 nm. The rate constants for formation of the 500 nm bands for the mutant enzymes are equal to or greater than those of wild-type Trpase, indicating that Asp-133 and His-458 do not play a role in the formation of quinonoid intermediates. In constrast to wild-type and H458A mutant Trpase, D133A mutant Trpase forms an intermediate from S-ethyl-L-Cys that absorbs at 345 nm, and is likely to be an alpha-aminoacrylate. Crystals of D133A and H458A mutant Trpase bind amino acids with similar affinity as the proteins in solution, except for L-Ala, which binds to D133A mutant Trpase crystals about 20-fold stronger than in solution. These results suggest that Asp-133 and His-458 play an important role in the elimination reaction of L-Trp. Asp-133 likely forms a hydrogen bond directly to the indole NH of the substrate, while His-458 probably is hydrogen bonded to Asp-133.     

Molecular modeling and site-directed mutagenesis define the catalytic motif in human gamma -glutamyl hydrolase

Human gamma-glutamyl hydrolase (hGH) is a central enzyme in folyl and antifolylpoly-gamma-glutamate metabolism, which functions by catalyzing the cleavage of the gamma-glutamyl chain of substrates. We previously reported that Cys-110 is essential for activity. Using the sequence of hGH as a query, alignment searches of protein data bases were made using the SSearch and TPROBE programs. Significant similarity was found between hGH and the glutamine amidotransferase type I domain of Escherichia coli carbamoyl phosphate synthetase. The resulting hypothesis is that the catalytic fold of hGH is similar to the folding of this domain in carbamoyl phosphate synthetase. This model predicts that Cys-110 of hGH is the active site nucleophile and forms a catalytic triad with residues His-220 and Glu-222. The hGH mutants C110A, H220A, and E222A were prepared. Consistent with the model, mutants C110A and H220A were inactive. However, the V(max) of the E222A hGH mutant was reduced only 6-fold relative to the wild-type enzyme. The model also predicted that His-171 in hGH may be involved in substrate binding. The H171N hGH mutant was found to have a 250-fold reduced V(max). These studies to determine the catalytic mechanism begin to define the three dimensional interactions of hGH with poly-gamma-glutamate substrates.     

Alpha-amino-beta-carboxymuconic-epsilon-semialdehyde decarboxylase (ACMSD) is a new member of the amidohydrolase superfamily

The enzymatic activity of Pseudomonas fluorescens alpha-amino-beta-carboxymuconic-epsilon-semialdehyde decarboxylase (ACMSD) is critically dependent on a transition metal ion [Li, T., Walker, A. L., Iwaki, H., Hasegawa, Y., and Liu, A. (2005) J. Am. Chem. Soc. 127, 12282-12290]. Sequence analysis in this study further suggests that ACMSD belongs to the amidohydrolase superfamily, whose structurally characterized members comprise a catalytically essential metal cofactor. To identify ACMSD's metal ligands and assess their functions in catalysis, a site-directed mutagenesis analysis was conducted. Alteration of His-9, His-177, and Asp-294 resulted in a dramatic loss of enzyme activity, substantial reduction of the metal-binding ability, and an altered metallocenter electronic structure. Thus, these residues are confirmed to be the endogenous metal ligands. His-11 is implicated in metal binding because of the strictly conserved HxH motif with His-9. Mutations at the 228 site yielded nearly inactive enzyme variants H228A and H228E. The two His-228 mutant proteins, however, exhibited full metal-binding ability and a metal center similar to that of the wild-type enzyme as shown by EPR spectroscopy. Kinetic analysis on the mutants indicates that His-228 is a critical catalytic residue along with the metal cofactor. Since the identified metal ligands and His-228 are present in all known ACMSD sequences, it is likely that ACMSD proteins from other organisms contain the same cofactor and share similar catalytic mechanisms. ACMSD is therefore the first characterized member in the amidohydrolase superfamily that represents a C-C breaking activity.     

A putative glutathione-binding site in T4 glutaredoxin investigated by site-directed mutagenesis

A glutathione monomer has been docked into the active site cleft of T4 glutaredoxin (previously called T4 thioredoxin) using molecular graphics. The central part of the cleft is formed by the side chain of Tyr-16 on one side and the residues Thr-64, Met-65, and Pro-66 on the other. The entire glutathione molecule fits well into the cleft. A cis-peptide bond between the residues Met-65 and Pro-66 allows glutathione to bind in an anti-parallel fashion to residues 64-66. Hydrogen bonds can be formed between Met-65 and the glutathione cysteine. This binding positions the glutathione sulfur atom ideally for reaction with the glutaredoxin disulfide. In the model, glutathione can form a hydrogen bond to the hydroxyl group of Tyr-16. Charged interactions at opposite ends of the binding cleft are provided by His-12 and Asp-80. The negatively charged alpha-carboxyl group of glutathione may interact with a positive helix dipole of the protein. Fifteen mutant T4 glutaredoxins have been produced and assayed for glutathione binding by determining thioltransferase activity. Mutant proteins with substitutions in the sides of the cleft (Tyr-16, Pro-66) exhibited the most marked decreases in thioltransferase activity. Mutation of His-12 to a serine decreases the catalytic efficiency whereas substitution of Asp-80 by serine increases the catalytic efficiency. A double mutant, D80S;H12S, has much less affinity for glutathione than either single mutant. Substitution of Cys-14 produces an inactive protein, whereas C17S retains some thioltransferase activity.     

Elucidation of the heme binding site of heme-regulated eukaryotic initiation factor 2alpha kinase and the role of the regulatory motif in heme sensing by spectroscopic and catalytic studies of mutant proteins

Heme-regulated eukaryotic initiation factor 2alpha (eIF2alpha) kinase (HRI) functions in response to the heme iron concentration. At the appropriate heme iron concentrations under normal conditions, HRI function is suppressed by binding of the heme iron. Conversely, upon heme iron shortage, HRI autophosphorylates and subsequently phosphorylates the substrate, eIF2alpha, leading to the termination of protein synthesis. The molecular mechanism of heme sensing by HRI, including identification of the specific binding site, remains to be established. In the present study we demonstrate that His-119/His-120 and Cys-409 are the axial ligands for the Fe(III)-protoporphyrin IX complex (hemin) in HRI, based on spectral data on site-directed mutant proteins. Cys-409 is part of the heme-regulatory Cys-Pro motif in the kinase domain. A P410A full-length mutant protein displayed loss of heme iron affinity. Surprisingly, inhibitory effects of the heme iron on catalysis and changes in the heme dissociation rate constants in full-length His-119/His-120 and Cys-409 mutant proteins were marginally different to wild type. In contrast, heme-induced inhibition of Cys-409 mutants of the isolated kinase domain and N-terminal-truncated proteins was substantially weaker than that of the full-length enzyme. A pulldown assay disclosed heme-dependent interactions between the N-terminal and kinase domains. Accordingly, we propose that heme regulation is induced by interactions between heme and the catalytic domain in conjunction with global tertiary structural changes at the N-terminal domain that accompany heme coordination and not merely by coordination of the heme iron with amino acids on the protein surface.