Characterization of cysteine residues of glutathione S-transferase P: evidence for steric hindrance of substrate binding by a bulky adduct to cysteine 47.

Glutathione S-transferase P (GST-P) lost the enzymatic activity by 7-fluoro-4-sulfamoyl-2, 1, 3-benzodiazole (ABD-F), a thiol-group chemical modifier, but did not by methylmethanethiol-sulfonate. Both ABD-F and methylmethanethiolsulfonate reacted with Cys47 and Cys101. These two cysteine residues were site-directedly mutated with serine residues. Only the Cys101Ser lost the enzymatic activity by the treatment of ABD-F. On carbon 13 NMR experiments, a NMR signal of S-[13C]CH3 adduct to Cys47 did not show any change by the addition of S-hexylglutathione. These facts revealed that Cys47 did not locate at the active site, and a bulky adduct to Cys47 hindered the binding of substrates to the active site.     

Ser(214) is crucial for substrate binding to serine proteases

Highly conserved amino acids that form crucial structural elements of the catalytic apparatus can be used to account for the evolutionary history of serine proteases and the cascades into which they are organized. One such evolutionary marker in chymotrypsin-like proteases is Ser(214), located adjacent to the active site and forming part of the primary specificity pocket. Here we report the mutation of Ser(214) in thrombin to Ala, Thr, Cys, Asp, Glu, and Lys. None of the mutants seriously compromises active site catalytic function as measured by the kinetic parameter k(cat). However, the least conservative mutations result in large increases in K(m) because of lower rates of substrate diffusion into the active site. Therefore, the role of Ser(214) is to promote the productive formation of the enzyme-substrate complex. The S214C mutant is catalytically inactive, which suggests that during evolution the TCN-->AGY codon transitions for Ser(214) occurred through Thr intermediates.     

Single base mutation in alpha 5(IV) collagen chain gene converting a conserved cysteine to serine in Alport syndrome.

We have identified a point mutation in the type IV collagen alpha 5 chain gene (COL4A5) in Alport syndrome. Variant PstI (Barker et al., 1990, Science 248, 1224-1227), and BglII restriction sites with complete linkage with the Alport phenotype have been found in the 3' end of the COL4A5 gene in the large Utah Kindred P. The approximate location of the variant sites was determined by restriction enzyme mapping, after which this region of the gene (1028 bp) was amplified with the polymerase chain reaction (PCR) from DNA of normal and affected individuals for sequencing analysis. The PCR products showed the absence or presence of the variant PstI and BglII sites in DNA from normal and affected individuals, respectively. DNA sequencing revealed a single base change in exon 3 (from the 3' end) in DNA from affected individuals, changing the TGT codon of cysteine to the TCT codon for serine. This single base mutation also generated new restriction sites for PstI and BglII. The mutation involves a cysteine residue that has remained conserved in the carboxyl-end noncollagenous domain (NC domain) of all known type IV collagen alpha chains from Drosophila to man. It is presumably crucial for maintaining the right conformation of the NC domain, which is important for both triple-helix formation and the formation of intermolecular cross-links of type IV collagen molecules.     

Identification of two serine residues involved in catalysis by fatty acid amide hydrolase.

Fatty acid amide hydrolase is an integral membrane protein that hydrolyzes a novel and growing class of neuromodulatory fatty acid molecules, including anandamide, 2-arachidonyl glycerol, and oleamide. This activity is inhibited by serine and cysteine reactive agents, suggesting that the active site contains a serine or cysteine residue. Therefore serine and cysteine residues were mutated to alanine and the effects on activity were determined. Mutants were prepared using site-directed mutagenesis methods and expressed in COS-7 cells. Serine mutations S217A and S241A completely abolished enzymatic activity. Mutants S152A and C249A had no effect on activity, while S218A showed a slight decrease in activity. To confirm these results biochemically, the mutant enzymes were reacted with the irreversible inhibitor [(14)C]-diisopropyl fluorophosphate. All of the mutants except S217A and S241A were labeled. We therefore confirm that fatty acid amide hydrolase is a serine hydrolase and propose that both Ser-217 and Ser-241 are essential for enzyme activity.     

Requirements for the mitochondrial import and localization of dihydroorotate dehydrogenase.

In animals, dihydroorotate dehydrogenase (DHODH) is a mitochondrial protein that carries out the fourth step in de novo pyrimidine biosynthesis. Because this is the only enzyme of this pathway that is localized to mitochondria and because the enzyme is cytosolic in some bacteria and fungi, we carried out studies to understand the mode of targeting of animal DHODH and its submitochondrial localization. Analysis of fractionated rat liver mitochondria revealed that DHODH is an integral membrane protein exposed to the intermembrane space. In vitro-synthesized Drosophila, rat and human DHODH proteins were efficiently imported into the intermembrane space of isolated yeast mitochondria. Import did not alter the size of the in vitro synthesized protein, nor was there a detectable size difference when compared to the DHODH protein found in vivo. Thus, there is no apparent proteolytic processing of the protein during import either in vitro or in vivo. Import of rat DHODH into isolated yeast mitochondria required inner membrane potential and was at least partially dependent upon matrix ATP, indicating that its localization uses the well described import machinery of the mitochondrial inner membrane. The DHODH proteins of animals differ from the cytosolic proteins found in some bacteria and fungi by the presence of an N-terminal segment that resembles mitochondrial-targeting presequences. Deletion of the cationic portion of this N-terminal sequence from the rat DHODH protein blocked its import into isolated yeast mitochondria, whereas deletion of the adjacent hydrophobic segment resulted in import of the protein into the matrix. Thus, the N-terminus of the DHODH protein contains a bipartite signal that governs import and correct insertion into the mitochondrial inner membrane.     

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a specific substrate of yeast metacaspase

Yeast metacaspase (Yca1p) is required for the execution of apoptosis upon a wide range of stimuli. However, the specific degradome of this yeast protease has not been unraveled so far. By combining different methodologies described as requisites for a protein to be considered a protease substrate, such as digestome analysis, cleavage of recombinant GAPDH by metacaspase and evaluation of protein levels in vivo, we show that upon H(2)O(2)-induced apoptosis, the metabolic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a specific target of metacaspase. Nitric oxide (NO) signaling, which mediates H(2)O(2)-induced apoptosis, is required for metacaspase specific GAPDH cleavage. In conclusion, in this work we identified GAPDH as the first direct yeast metacaspase substrate described so far. Although mammalian caspases and yeast metacaspase apparently have distinct target cleavage sites, GAPDH arises as a common substrate for these proteases.     

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.     

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.     

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.     

The cysteine synthase complex from plants. Mitochondrial serine acetyltransferase from Arabidopsis thaliana carries a bifunctional domain for catalysis and protein-protein interaction.

Serine acetyltransferase (SAT) catalyzes the rate-limiting step of cysteine biosynthesis in bacteria and plants and functions in association with O-acetylserine (thiol) lyase (OAS-TL) in the cysteine synthase complex. Very little is known about the structure and catalysis of SATs except that they share a characteristic C-terminal hexapeptide-repeat domain with a number of enzymatically unrelated acyltransferases. Computational modeling of this domain was performed for the mitochondrial SAT isoform from Arabidopsis thaliana, based on crystal structures of bacterial acyltransferases. The results indicate a left-handed parallel beta-helix consisting of beta-sheets alternating with turns, resulting in a prism-like structure. This model was challenged by site-directed mutagenesis and tested for a suspected dual function of this domain in catalysis and hetero-oligomerization. The bifunctionality of the SAT C-terminus in transferase activity and interaction with OAS-TL is demonstrated and discussed with respect to the putative role of the cysteine synthase complex in regulation of cysteine biosynthesis.     

Lipoprotein (a) is a substrate for factor XIIIa and tissue transglutaminase.

The mechanisms which mediate deposition of lipoprotein (a) (Lp(a)), an atherogenic lipoprotein particle, onto the vessel wall and cell surfaces are unknown. An irreversible deposition of Lp(a) may require the presence of enzymes that catalyze its binding to surface-oriented structures. Transglutaminases catalyze cross-linking of proteins as well as incorporation of primary amines into protein substrates. We studied whether tissue transglutaminase and/or activated Factor XIII (plasma derived or recombinant FXIIIa) incorporate primary amines into Lp(a). In the presence of Ca2+, Factor XIIIa and tissue transglutaminase catalyze incorporation of monodansylcadaverine or [14C]putrescine into purified Lp(a) in a specific and time-dependent manner. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that monodansylcadaverine became incorporated into the apo(a) portion of Lp(a). Lp(a) purified from five different donors showing different apo(a) phenotypes were substrates for tissue transglutaminases (TG). Western blot analysis confirmed that apo(a) was the major monodansylcadaverine carrying protein moiety of Lp(a). Tissue TG also extensively cross-linked the apo(a) portion of the Lp(a) particle. Characterization of the specificity of tissue TG showed that fibronectin, alpha 2-plasmin inhibitor, and apo(a) could be readily labeled with monodansylcadaverine by tissue TG, but other proteins including low density lipoprotein, IgG, alpha 1-proteinase inhibitor, and albumin showed poor or no reactivity. Direct comparison of Lp(a) with low density lipoprotein showed that apoB 100 was a poor substrate for transglutaminases. Recombinant apolipoprotein (a) proved to be an excellent substrate for TGs in that 1 mol of recombinant apolipoprotein (a) incorporated as much as 15 mol of [14C]putrescine, which corresponded to five times the amount of amine incorporated into Lp(a). The susceptibility of Lp(a) to transglutaminases suggests a mechanism whereby the interaction of Lp(a) with surface receptors and other surface oriented structures could be enzymatically altered.     

Conserved cysteine residues determine substrate specificity in a novel As(III) S‐adenosylmethionine methyltransferase from Aspergillus fumigatus

Methylation of inorganic arsenic is a central process in the organoarsenical biogeochemical cycle. Members of every kingdom have ArsM As(III) S‐adenosylmethionine (SAM) methyltransferases that methylates inorganic As(III) into mono‐ (MAs(III)), di‐ (DMAs(III)) and tri‐ (TMAs(III)) methylarsenicals. Every characterized ArsM to date has four conserved cysteine residues. All four cysteines are required for methylation of As(III) to MAs(III), but methylation of MAs(III) to DMAs(III) requires only the two cysteines closest to the C‐terminus. Fungi produce volatile and toxic arsines, but the physiological roles of arsenic methylation and the biochemical basis is unknown. Here they demonstrate that most fungal species have ArsM orthologs with only three conserved cysteine residues. The genome of Aspergillus fumigatus has four arsM genes encoding ArsMs with only the second, third and fourth conserved cysteine residues. AfArsM1 methylates MAs(III) but not As(III). Heterologous expression of AfarsM1 in an Escherichia coli conferred resistance to MAs(III) but not As(III). The existence of ArsMs with only three conserved cysteine residues suggest that the ability to methylate MAs(III) may be an evolutionary step toward enzymes capable of methylating As(III), the result of a loss of function mutation in organisms with infrequent exposure to inorganic As(III) or as a resistance mechanism for MAs(III).     

Optimizing the Michaelis complex of trimethylamine dehydrogenase: identification of interactions that perturb the ionization of substrate and facilitate catalysis with trimethylamine base.

Recent evidence from isotope studies supports the view that catalysis by trimethylamine dehydrogenase (TMADH) proceeds from a Michaelis complex involving trimethylamine base and not, as thought previously, trimethylammonium cation. In native TMADH reduction of the flavin by substrate (perdeuterated trimethylamine) is influenced by two ionizations in the Michaelis complex with pK(a) values of 6.5 and 8.4; maximal activity is realized in the alkaline region. The latter ionization has been attributed to residue His-172 and, more recently, the former to the ionization of substrate itself. In the Michaelis complex, the ionization of substrate (pK(a) approximately 6.5 for perdeuterated substrate) is perturbed by approximately -3.3 to -3.6 pH units compared with that of free trimethylamine (pK(a) = 9.8) and free perdeuterated trimethylamine (pK(a) = 10.1), respectively, thus stabilizing trimethylamine base by approximately 2 kJ mol(-1). We show, by targeted mutagenesis and stopped-flow studies that this reduction of the pK(a) is a consequence of electronic interaction with residues Tyr-60 and His-172, thus these two residues are key for optimizing catalysis in the physiological pH range. We also show that residue Tyr-174, the remaining ionizable group in the active site that we have not targeted previously by mutagenesis, is not implicated in the pH dependence of flavin reduction. Formation of a Michaelis complex with trimethylamine base is consistent with a mechanism of amine oxidation that we advanced in our previous computational and kinetic studies which involves nucleophilic attack by the substrate nitrogen atom on the electrophilic C4a atom of the flavin isoalloxazine ring. Stabilization of trimethylamine base in the Michaelis complex over that in free solution is key to optimizing catalysis at physiological pH in TMADH, and may be of general importance in the mechanism of other amine dehydrogenases that require the unprotonated form of the substrate for catalysis.     

General base catalysis in a glutamine for histidine mutant at position 51 of human liver alcohol dehydrogenase

On the basis of the three-dimensional structure of horse liver alcohol dehydrogenase determined by X-ray crystallography, His 51 has been proposed to act as a general base during catalysis by abstracting a proton from the alcohol substrate. A hydrogen-bonding system (proton relay system) connecting the alcohol substrate and His 51 has been proposed to mediate proton transfer. We have mutated His 51 to Gln in the homologous human liver beta 1 beta 1 alcohol dehydrogenase isoenzyme which is expected to have a similar proton relay system. The mutation resulted in an about 6-fold drop in V/Kb (Vmax for ethanol oxidation divided by Km for ethanol) at pH 7.0 and a 12-fold drop at pH 6.5. V/Kb could be restored completely or partially by the presence of high concentrations of glycylglycine, glycine, and phosphate buffers. A Br?nsted plot of the effect on V/Kb versus the pKa of these bases plus H2O and OH- was linear. Only secondary or tertiary amine buffers differed from linearity, presumably due to steric hindrance. These results suggest that His 51 acts as a general base catalyst during alcohol oxidation in the wild-type enzyme and can be functionally replaced in the mutant enzyme by general base catalysts present in the solvent. Steady-state kinetic constants for NAD+ and the trifluoroethanol inhibition patterns were similar between the wild-type and the mutant enzyme. Differences in the inhibition constants (Ki) of caprate and trifluoroethanol below pH 7.8 and in the pH dependence of Ki can be explained by the substitution of neutral Gln for positively charged His.     

Deoxyhypusine hydroxylase is a Fe(II)-dependent, HEAT-repeat enzyme. Identification of amino acid residues critical for Fe(II) binding and catalysis [corrected

Deoxyhypusine hydroxylase (DOHH) catalyzes the final step in the post-translational synthesis of hypusine (N(epsilon)-(4-amino-2-hydroxybutyl)lysine) in eIF5A. DOHH is a HEAT-repeat protein with eight tandem helical hairpins in a symmetrical dyad. It contains two potential iron coordination sites (one on each dyad) composed of two strictly conserved His-Glu motifs. The purified human recombinant DOHH was a mixture of active holoenzyme containing 2 mol of iron/mol of DOHH and inactive metal-free apoenzyme. The two species could be distinguished by their different mobilities upon native gel electrophoresis. The DOHH apoenzyme exhibited markedly reduced levels of iron and activity. DOHH activity could be restored only by the addition of Fe2+ to the apoenzyme but not by other metals including Cd2+,Co2+,Cr2+,Cu2+,Mg2+,Mn2+,Ni2+, and Zn2+. The role of the strictly conserved His-Glu residues was evaluated by site-directed mutagenesis. Substitution of any single amino acid in the four His-Glu motifs with alanine abolished the enzyme activity. Of these eight alanine substitutions, six, including H56A, H89A, E90A, H207A, H240A, and E241A, caused a severe reduction in the iron content. Our results provide strong evidence that Fe(II) is the active-site-bound metal critical for DOHH catalysis and that the strictly conserved His-Glu motifs are essential for iron binding and catalysis. Furthermore, the iron to DOHH stoichiometry and dependence of iron binding on each of the four conserved His-Glu motifs suggest a binuclear iron mediated reaction mechanism, distinct from that of other Fe(II)-dependent protein hydroxylases, such as prolyl 4-hydroxylase or lysyl hydroxylases.     

Identification of a novel gene of pyrimidine nucleotide biosynthesis, pyrDII, that is required for dihydroorotate dehydrogenase activity in Bacillus subtilis.

An in-frame deletion in the coding region of a gene of previously unidentified function (which is called orf2 and which we propose to rename pyrDII) in the Bacillus subtilis pyr operon led to pyrimidine bradytrophy, markedly reduced dihydroorotate dehydrogenase activity, and derepressed levels of other enzymes of pyrimidine biosynthesis. The deletion mutation was not corrected by a plasmid encoding pyrDI, the previously identified gene encoding dihydroorotate dehydrogenase, but was complemented by a plasmid encoding pyrDII. We propose that pyrDII encodes a protein subunit of dihydroorotate dehydrogenase that catalyzes electron transfer from the pyrDI-encoded subunit to components of the electron transport chain.     

The serpin SQN-5 is a dual mechanistic-class inhibitor of serine and cysteine proteinases

SQN-5 is a mouse serpin that is highly similar to the human serpins SCCA1 (SERPINB3) and SCCA2 (SERPINB4). Previous studies characterizing the biochemical activity of SQN-5 showed that this serpin, like SCCA2, inhibited the chymotrypsin-like enzymes mast cell chymase and cathepsin G. Using an expanded panel of papain-like cysteine proteinases, we now show that SQN-5, like SCCA1, inhibited cathepsins K, L, S, and V but not cathepsin B or H. These interactions were characterized by stoichiometries of inhibition that were nearly 1:1 and second-order rate constants of >10(4) M(-1) s(-1). Reactive site loop (RSL) cleavage analysis showed that SQN-5 employed different reactive centers to neutralize the serine and cysteine proteinases. To our knowledge, this is the first serpin that serves as a dual inhibitor of both chymotrypsin-like serine and the papain-like cysteine proteinases by employing an RSL-dependent inhibitory mechanism. The ability of serpins to inhibit both serine and/or papain-like cysteine proteinases may not be a recent event in mammalian evolution. Phylogenetic studies suggested that the SCCA and SQN genes evolved from a common ancestor approximately 250-280 million years ago. When the fact that mammals and birds diverged approximately 310 million years ago is considered, an ancestral SCCA/SQN-like serpin with dual inhibitory activity may be present in many mammalian genomes.     

Proton transfer to residues of basic pK(a) during catalysis by carbonic anhydrase.

The maximal velocity in the hydration of CO(2) catalyzed by the carbonic anhydrases in well-buffered solutions is limited by an intramolecular proton transfer from zinc-bound water to acceptor groups of the enzyme and hence to buffer in solution. Stopped-flow spectrophotometry was used to accumulate evidence that this maximal velocity is affected by residues of basic pK(a), near 8 to above 9, in catalysis of the hydration of CO(2) by carbonic anhydrases III, IV, V, and VII. A mutant of carbonic anhydrase II containing the replacement His-64-->Ala, which removes the prominent histidine proton shuttle (with pK(a) near 7), allows better observation of these basic groups. We suggest this feature of catalysis is general for the human and animal carbonic anhydrases and is due to residues of basic pK(a), predominantly lysines and tyrosines more distant from the zinc than His-64, that act as proton acceptors. These groups supplement the well-studied proton transfer from zinc-bound water to His-64 in the most efficient of the carbonic anhydrases, isozymes II, IV, and VII.