Crystal structure of the his-tagged saccharopine reductase from Saccharomyces cerevisiae at 1.7-Å resolution

The three-dimensional structure of the saccharopine reductase enzyme from the budding yeast Saccharomyces cerevisiae was determined to 1.7-A resolution in the apo form by using molecular replacement. The enzyme monomer consists of three domains: domain I is a variant of the Rossmann fold, domain II folds into a alpha/beta structure containing a mixed seven-stranded beta-sheet as the central core, and domain III has an all-helical fold. Comparative fold alignment with the enzyme from Magnaporthe grisea suggests that domain I binds to NADPH, and domain II binds to saccharopine and is involved in dimer formation. Domain III is involved in closing the active site of the enzyme once substrates are bound. Structural comparison of the saccharopine reductase enzymes from S. cerevisiae and M. grisea indicates that domain II has the highest number of conserved residues, suggesting that it plays an important role in substrate binding and in spatially orienting domains I and III.     

Structural studies of the final enzyme in the alpha-aminoadipate pathway-saccharopine dehydrogenase from Saccharomyces cerevisiae

The 1.64 A structure of the apoenzyme form of saccharopine dehydrogenase (SDH) from Saccharomyces cerevisiae shows the enzyme to be composed of two domains with similar dinucleotide binding folds with a deep cleft at the interface. The structure reveals homology to alanine dehydrogenase, despite low primary sequence similarity. A model of the ternary complex of SDH, NAD, and saccharopine identifies residues Lys77 and Glu122 as potentially important for substrate binding and/or catalysis, consistent with a proton shuttle mechanism. Furthermore, the model suggests that a conformational change is required for catalysis and that residues Lys99 and Asp281 may be instrumental in mediating this change. Analysis of the crystal structure in the context of other homologous enzymes from pathogenic fungi and human sources sheds light into the suitability of SDH as a target for antimicrobial drug development.     

X-ray structure of 5-aminolevulinic acid dehydratase from Escherichia coli complexed with the inhibitor levulinic acid at 2.0 A resolution

5-Aminolevulinic acid dehydratase (ALAD), an early enzyme of the tetrapyrrole biosynthesis pathway, catalyzes the dimerization of 5-aminolevulinic acid to form the pyrrole, porphobilinogen. ALAD from Escherichia coli is shown to form a homo-octameric structure with 422 symmetry in which each subunit adopts the TIM barrel fold with a 30-residue N-terminal arm. Pairs of monomers associate with their arms wrapped around each other. Four of these dimers interact, principally via their arm regions, to form octamers in which each active site is located on the surface. The active site contains two lysine residues (195 and 247), one of which (Lys 247) forms a Schiff base link with the bound substrate analogue, levulinic acid. Of the two substrate binding sites (referred to as A and P), our analysis defines the residues forming the P-site, which is where the first ALA molecule to associate with the enzyme binds. The carboxyl group of the levulinic acid moiety forms hydrogen bonds with the side chains of Ser 273 and Tyr 312. In proximity to the levulinic acid is a zinc binding site formed by three cysteines (Cys 120, 122, and 130) and a solvent molecule. We infer that the second substrate binding site (or A-site) is located between the triple-cysteine zinc site and the bound levulinic acid moiety. Two invariant arginine residues in a loop covering the active site (Arg 205 and Arg 216) appear to be appropriately placed to bind the carboxylate of the A-site substrate. Another metal binding site, close to the active site flap, in which a putative zinc ion is coordinated by a carboxyl and five solvent molecules may account for the activating properties of magnesium ions.     

Variation of (2''-5'')oligo(adenylate) synthetase activity during rat-liver regeneration

The inhibitory effect of fructose 2,6-biphosphate on fructose 1,6-bisphosphatase was reinvestigated in order to solve the apparent contradiction between competition with the substrate and the synergism with AMP, a strictly noncompetitive inhibitor. The effect of fructose 2,6-bisphosphate was compared to that of other ligands of the enzyme, which, like the substrate and methyl (alpha + beta)fructofuranoside 1,6-bisphosphate bind to the active site or which, like AMP, bind to an allosteric site. An increase in temperature or pH, or the presence of sulfosalicylate, lithium or higher concentrations of magnesium as well as partial proteolysis by subtilisin increased [I]0.5 for fructose 2,6-bisphosphate and AMP without affecting Km. With the exception of the pH change, all these conditions were also without effect on the affinity of the enzyme for the competitive inhibitor, methyl (alpha + beta)fructofuranoside 1,6-bisphosphate. These observations can be explained by assuming that fructose 2,6-bisphosphate has no affinity for the active site of fructose 1,6-bisphosphatase but binds to an allosteric site which is different from the AMP site. Fructose 2,6-bisphosphate is therefore classified as an allosteric competitive inhibitor and a model is proposed which explains its synergism with AMP as well as the various cooperative effects.     

Lysine 274 is essential for fructose 2,6-bisphosphate inhibition of fructose-1,6-bisphosphatase.

Lysine 274 is conserved in all known fructose-1,6-bisphosphatase sequences. It has been implicated in substrate binding and/or catalysis on the basis of reactivity with pyridoxal phosphate as well as by x-ray crystallographic analysis. Lys274 of rat liver fructose-1,6-bisphosphatase was mutated to alanine by the polymerase chain reaction, and the T7-RNA polymerase-transcribed construct containing the mutant sequence was expressed in Escherichia coli. The mutant and wild-type forms of the enzyme were purified to homogeneity, and their specific activity, substrate dependence, and inhibition by fructose 2,6-bisphosphate and AMP were compared. While the mutant exhibited no change in maximal velocity, its Km for fructose 1,6-bisphosphate was 20-fold higher than that of the wild-type, and its Ki for fructose 2,6-bisphosphate was increased 1000-fold. Consistent with the unaltered maximal velocity, there were no apparent difference between the secondary structure of the wild-type and mutant enzyme forms, as measured by circular dichroism and ultraviolet difference spectroscopy. The Ki for the allosteric inhibitor AMP was only slightly increased, indicating that Lys274 is not directly involved in AMP inhibition. Fructose 2,6-bisphosphate potentiated AMP inhibition of both forms, but 500-fold higher concentrations of fructose 2,6-bisphosphate were needed to reduce the Ki for AMP for the mutant compared to the wild-type. However, potentiation of AMP inhibition of the Lys274----Ala mutant was evident at fructose 2,6-bisphosphate concentrations (approximately 100 microM) well below those that inhibited the enzyme, which suggests that fructose 2,6-bisphosphate interacts either with the AMP site directly or with other residues involved in the active site-AMP synergy. The results also demonstrate that although Lys274 is an important binding site determinant for sugar bisphosphates, it plays a more significant role in binding fructose 2,6-bisphosphate than fructose 1,6-bisphosphate, probably because it binds the 2-phospho group of the former while other residues bind the 1-phospho group of the substrate. It is concluded that the enzyme utilizes Lys274 to discriminate between its substrate and fructose 2,6-bisphosphate.     

Crystal structures of adenine phosphoribosyltransferase from Leishmania donovani.

The enzyme adenine phosphoribosyltransferase (APRT) functions to salvage adenine by converting it to adenosine-5-monophosphate (AMP). APRT deficiency in humans is a well characterized inborn error of metabolism, and APRT may contribute to the indispensable nutritional role of purine salvage in protozoan parasites, all of which lack de novo purine biosynthesis. We determined crystal structures for APRT from Leishmania donovani in complex with the substrate adenine, the product AMP, and sulfate and citrate ions that appear to mimic the binding of phosphate moieties. Overall, these structures are very similar to each other, although the adenine and AMP complexes show different patterns of hydrogen-bonding to the base, and the active site pocket opens slightly to accommodate the larger AMP ligand. Whereas AMP adopts a single conformation, adenine binds in two mutually exclusive orientations: one orientation providing adenine-specific hydrogen bonds and the other apparently positioning adenine for the enzymatic reaction. The core of APRT is similar to that of other phosphoribosyltransferases, although the adenine-binding domain is quite different. A C-terminal extension, unique to Leishmania APRTs, extends an extensive dimer interface by wrapping around the partner molecule. The active site involves residues from both subunits of the dimer, indicating that dimerization is essential for catalysis.     

Crystal structure of glycerophosphodiester phosphodiesterase (GDPD) from Thermoanaerobacter tengcongensis, a metal ion-dependent enzyme: insight into the catalytic mechanism

Glycerophosphodiester phosphodiesterase (GDPD; EC 3.1.4.46) catalyzes the hydrolysis of a glycerophosphodiester to an alcohol and glycerol 3-phosphate in glycerol metabolism. It has an important role in the synthesis of a variety of products that participate in many biochemical pathways. We report the crystal structure of the Thermoanaerobacter tengcongensis GDPD (ttGDPD) at 1.91 A resolution, with a calcium ion and glycerol as a substrate mimic coordinated at this calcium ion (PDB entry 2pz0). The ttGDPD dimer with an intermolecular disulfide bridge and two hydrogen bonds is considered as the potential functional unit. We used site-directed mutagenesis to characterize ttGDPD as a metal ion-dependent enzyme, identified a cluster of residues involved in substrate binding and the catalytic reaction, and we propose a possible general acid-base catalytic mechanism for ttGDPD. Superposing the active site with the homologous structure GDPD from Agrobacterium tumefaciens (PDB entry 1zcc), which binds a sulfate ion in the active site, the sulfate ion can represent the phosphate moiety of the substrate, simulating the binding mode of the true substrate of GDPD.     

Crystal structure of saccharopine reductase from Magnaporthe grisea, an enzyme of the alpha-aminoadipate pathway of lysine biosynthesis

BACKGROUND: The biosynthesis of the essential amino acid lysine in higher fungi and cyanobacteria occurs via the alpha-aminoadipate pathway, which is completely different from the lysine biosynthetic pathway found in plants and bacteria. The penultimate reaction in the alpha-aminoadipate pathway is catalysed by NADPH-dependent saccharopine reductase. We set out to determine the structure of this enzyme as a first step in exploring the structural biology of fungal lysine biosynthesis. RESULTS: We have determined the three-dimensional structure of saccharopine reductase from the plant pathogen Magnaporthe grisea in its apo form to 2.0 A resolution and as a ternary complex with NADPH and saccharopine to 2.1 A resolution. Saccharopine reductase is a homodimer, and each subunit consists of three domains, which are not consecutive in amino acid sequence. Domain I contains a variant of the Rossmann fold that binds NADPH. Domain II folds into a mixed seven-stranded beta sheet flanked by alpha helices and is involved in substrate binding and dimer formation. Domain III is all-helical. The structure analysis of the ternary complex reveals a large movement of domain III upon ligand binding. The active site is positioned in a cleft between the NADPH-binding domain and the second alpha/beta domain. Saccharopine is tightly bound to the enzyme via a number of hydrogen bonds to invariant amino acid residues. CONCLUSIONS: On the basis of the structure of the ternary complex of saccharopine reductase, an enzymatic mechanism is proposed that includes the formation of a Schiff base as a key intermediate. Despite the lack of overall sequence homology, the fold of saccharopine reductase is similar to that observed in some enzymes of the diaminopimelate pathway of lysine biosynthesis in bacteria. These structural similarities suggest an evolutionary relationship between two different major families of amino acid biosynthetic pathway, the glutamate and aspartate families.     

Crystal structure of sorbitol dehydrogenase

Sorbitol dehydrogenase (SDH) is a distant relative to the alcohol dehydrogenases (ADHs) with sequence identities around 20%. SDH is a tetramer with one zinc ion per subunit. We have crystallized rat SDH and determined the structure by molecular replacement using a tetrameric bacterial ADH as search object. The conformation of the bound coenzyme is extended and similar to NADH bound to mammalian ADH but the interactions with the NMN-part have several differences with those of ADH. The active site zinc coordination in SDH is significantly different than in mammalian ADH but similar to the one found in the bacterial tetrameric NADP(H)-dependent ADH of Clostridiim beijerinckii. The substrate cleft is significantly more polar than for mammalian ADH and a number of residues are ideally located to position the sorbitol molecule in the active site. The SDH molecule can be considered to be a dimer of dimers, with subunits A-B and C-D, where the dimer interactions are similar to those in mammalian ADH. The tetramers are composed of two of these dimers, which interact with their surfaces opposite the active site clefts, which are accessible on the opposite side. In contrast to the dimer interactions, the tetramer-forming interactions are small with only few hydrogen bonds between side-chains.     

Crystal structures of the catalytic domain of phosphodiesterase 4B complexed with AMP, 8-Br-AMP, and rolipram

Phosphodiesterase catalyzes the hydrolysis of the intracellular second messenger 3',5'-cyclic AMP (cAMP) into the corresponding 5'-nucleotide. Phosphodiesterase 4 (PDE4), the major cAMP-specific PDE in inflammatory and immune cells, is an attractive target for the treatment of asthma and COPD. We have determined crystal structures of the catalytic domain of PDE4B complexed with AMP (2.0 A), 8-Br-AMP (2.13 A) and the potent inhibitor rolipram (2.0 A). All the ligands bind in the same hydrophobic pocket and can interact directly with the active site metal ions. The identity of these metal ions was examined using X-ray anomalous difference data. The structure of the AMP complex confirms the location of the catalytic site and allowed us to speculate about the detailed mechanism of catalysis. The high-resolution structures provided the experimental insight into the nucleotide selectivity of phosphodiesterase. 8-Br-AMP binds in the syn conformation to the enzyme and demonstrates an alternative nucleotide-binding mode. Rolipram occupies much of the AMP-binding site and forms two hydrogen bonds with Gln443 similar to the nucleotides.     

The regulation of the interaction between F-actin and muscle fructose 1,6-bisphosphatase

Interaction between rabbit muscle fructose 1,6-bisphosphatase (FBPase) and rabbit muscle F-actin results in heterologous complex formation [A. Gizak, D. Rakus, A. Dzugaj, Histol. Histopathol. 18 (2003) 135]. Calculated on the basis of co-sedimentation-binding experiments and ELISA assay-binding constant (Ka) revealed that FBPase binds to F-actin with Ka equal to 7.4 x 10(4) M(-1). The binding is down-regulated by ligands interacting with the FBPase active site (fructose 6-phosphate, fructose 1,6-bisphosphate, fructose 2,6-bisphosphate) and with the FBPase allosteric inhibitory site (AMP). The binding and the kinetic data suggests that FBPase may bind F-actin using a bipartite motif which includes the amino acids residues involved in the binding of the substrate as well as of the allosteric inhibitor of the enzyme. The in situ co-localization experiment, in which FBPase was diffused into skinned muscle fibres pre-incubated with phalloidin (polymeric actin-interacting toxin), has shown that FBPase binds predominantly to the region of the Z-line.     

Crystal structure of sorbitol dehydrogenase

Sorbitol dehydrogenase (SDH) is a distant relative to the alcohol dehydrogenases (ADHs) with sequence identities around 20%. SDH is a tetramer with one zinc ion per subunit. We have crystallized rat SDH and determined the structure by molecular replacement using a tetrameric bacterial ADH as search object. The conformation of the bound coenzyme is extended and similar to NADH bound to mammalian ADH but the interactions with the NMN-part have several differences with those of ADH. The active site zinc coordination in SDH is significantly different than in mammalian ADH but similar to the one found in the bacterial tetrameric NADP(H)-dependent ADH of Clostridiim beijerinckii. The substrate cleft is significantly more polar than for mammalian ADH and a number of residues are ideally located to position the sorbitol molecule in the active site. The SDH molecule can be considered to be a dimer of dimers, with subunits A–B and C–D, where the dimer interactions are similar to those in mammalian ADH. The tetramers are composed of two of these dimers, which interact with their surfaces opposite the active site clefts, which are accessible on the opposite side. In contrast to the dimer interactions, the tetramer-forming interactions are small with only few hydrogen bonds between side-chains.     

Structural and mechanistic analysis of a novel class of shikimate dehydrogenases: evidence for a conserved catalytic mechanism in the shikimate dehydrogenase family

Shikimate dehydrogenase (SDH) catalyzes the reversible NADPH-dependent reduction of 3-dehydroshikimate to shikimate. This reaction represents the fourth step of the shikimate pathway, the essential route for the biosynthesis of the aromatic amino acids in plants, fungi, bacteria, and apicomplexan parasites. The absence of this pathway in animals makes it an attractive target for herbicides and antimicrobials. At least four functionally distinct enzyme classes, AroE, YdiB, SDH-like (SdhL), and AroE-like1 (Ael1), utilize shikimate as a substrate in vitro and form the SDH family. Crystal structures have been determined for AroE, YdiB, and SdhL. In this study, we have determined the first representative crystal structure of an Ael1 enzyme. We demonstrate that Ael1 shares a similar overall structure with the other members of the SDH family. This high level of structural conservation extends to the active sites of the enzymes. In particular, an ionizable active site lysine and aspartate are present in all SDH homologues. Two distinct biochemical roles have been reported for this Lys-Asp pair: as binding residues in YdiB and as a catalytic dyad in AroE and SdhL. Here, we establish that the residues function as a catalytic dyad in Ael1 and, interestingly, in at least one YdiB homologue. The conservation of three-dimensional fold, active site architecture, and catalytic mechanism among members of the SDH family will facilitate the design of drugs targeting the shikimate pathway.     

Inhibition of alpha-glucan phosphorylase by alpha-D-glucopyranosyl fluoride.

Alpha-D-Glucopyranosyl fluoride was found to inhibit strongly the action of alpha-glucan phosphorylase b[EC 2.4.1.1] from rabbit muscle, and that of the enzyme from potato tubers rather weakly. The inhibition is highly specific, being competitive with respect to glucose 1-phosphate and noncompetitive with respect to polysaccharide, during polysaccharide synthesis. In the reverse process, it is competitive with respect to Pi. These results have been explained by assuming that the inhibitor binds to the glucose 1-phosphate site of the enzyme, occupying both subsites which normally bind the glucosyl and phosphate moities of the substrate, but does not directly interact with the polysaccharide site. Based on this assumption, the dissociation constants of the enzyme-inhibitor and enzyme-polysaccharide-inhibitor complexes have been evaluated (0.43 and 0.20 mM for the muscle enzyme, respectively; 24 and 23 mM for the potato enzyme, respectively). Glucosyl fluoride also acts as a noncompetitive inhibitor with respect to AMP. A high concentration of AMP causes an inhibitory effect on the action of the muscle enzyme, the effect being menifested in the presence of glucosyl fluoride.     

Catalytic and regulatory site composition of yeast AMP deaminase by comparative binding and rate studies. Resolution of the cooperative mechanism

Yeast AMP deaminase is allosterically activated by ATP and MgATP and inhibited by GTP and PO4. The tetrameric enzyme binds 2 mol each of ATP, GTP, and PO4/subunit with Kd values of 8.4 +/- 4.0, 4.1 +/- 0.6, and 169 +/- 12 microM, respectively. At 0.7 M KCl, ATP binds to the enzyme, but no longer activates. Titration with coformycin 5'-monophosphate, a slow, tight-binding inhibitor, indicates a single catalytic site/subunit. ATP and GTP bind at regulatory sites distinct from the catalytic site and their binding is mutually exclusive. Inorganic phosphate competes poorly with ATP for the ATP sites (Kd = 20.1 +/- 4.1 mM). However, near-saturating ATP reduces the moles of phosphate bound per subunit to 1 PO4, which binds with a Kd = 275 +/- 22 microM. In the presence of ATP, PO4 cannot effectively compete with ATP for the nucleotide triphosphate sites. The PO4 which binds in the presence of ATP is competitive with AMP at the catalytic site since the Kd equals the kinetic inhibition constant for PO4. Initial reaction rate curves are a cooperative function of AMP concentration and activation by ATP is also cooperative. However, no cooperativity is observed in the binding of any of the regulator ligands and ATP binding and kinetic activation by ATP is independent of substrate analog concentration. Cooperativity in initial rate curves results, therefore, from altered rate constants for product formation from each (enzyme.substrate)n species and not from cooperative substrate binding. The traditional cooperative binding models of allosteric regulation do not apply to yeast AMP deaminase, which regulates catalytic activity by kinetic control of product formation. The data are used to estimate the rates of AMP hydrolysis under reported metabolite concentrations in yeast.     

Structural comparison of cytochromes P450 2A6, 2A13, and 2E1 with pilocarpine

Human xenobiotic-metabolizing cytochrome P450 (CYP) enzymes can each bind and monooxygenate a diverse set of substrates, including drugs, often producing a variety of metabolites. Additionally, a single ligand can interact with multiple CYP enzymes, but often the protein structural similarities and differences that mediate such overlapping selectivity are not well understood. Even though the CYP superfamily has a highly canonical global protein fold, there are large variations in the active site size, topology, and conformational flexibility. We have determined how a related set of three human CYP enzymes bind and interact with a common inhibitor, the muscarinic receptor agonist drug pilocarpine. Pilocarpine binds and inhibits the hepatic CYP2A6 and respiratory CYP2A13 enzymes much more efficiently than the hepatic CYP2E1 enzyme. To elucidate key residues involved in pilocarpine binding, crystal structures of CYP2A6 (2.4 ?), CYP2A13 (3.0 ?), CYP2E1 (2.35 ?), and the CYP2A6 mutant enzyme, CYP2A6?I208S/I300F/G301A/S369G (2.1 ?) have been determined with pilocarpine in the active site. In all four structures, pilocarpine coordinates to the heme iron, but comparisons reveal how individual residues lining the active sites of these three distinct human enzymes interact differently with the inhibitor pilocarpine.     

Distinct binding of cholesterol and 5beta-cholestane-3alpha,7alpha,12alpha-triol to cytochrome P450 27A1: evidence from modeling and site-directed mutagenesis studies

Cytochrome P450 27A1 (P450 27A1 or CYP27A1) is an important enzyme that participates in different pathways of cholesterol degradation as well as in the activation of vitamin D(3). Several approaches were utilized to investigate how two physiological substrates, cholesterol and 5beta-cholestane-3alpha,7alpha,12alpha-triol, interact with CYP27A1. The enzyme active site was first probed spectrally by assessing binding of the two substrates and five substrate analogues followed by computer modeling and site-directed mutagenesis. The computer models suggest that the spatial positions and orientations of cholesterol and 5beta-cholestane-3alpha,7alpha,12alpha-triol are different in the enzyme active site. As a result, some of the active site residues interact with both substrates, although they are situated differently relative to each steroid, and some residues bind only one substrate. Mutation of the overlapping substrate-contact residues (W100, H103, T110, M301C, V367, I481, and V482) affected CYP27A1 binding and enzyme activity in a substrate-dependent manner and allowed identification of several important side chains. T110 is proposed to interact with the 12alpha-hydroxyl of 5beta-cholestane-3alpha,7alpha,12alpha-triol, whereas V367 seems to be crucial for correct positioning of the cholesterol C26 methyl group and for regioselective hydroxylation of this substrate. Distinct binding of the CYP27A1 substrates may provide insight into why phenotypic manifestations of cerebrotendinous xanthomatosis, a disease associated with CYP27A1 deficiency, are so diverse.     

Structural and kinetic analyses of arginine residues in the active site of the acetate kinase from Methanosarcina thermophila

Acetate kinase catalyzes transfer of the gamma-phosphate of ATP to acetate. The only crystal structure reported for acetate kinase is the homodimeric enzyme from Methanosarcina thermophila containing ADP and sulfate in the active site (Buss, K. A., Cooper, D. C., Ingram-Smith, C., Ferry, J. G., Sanders, D. A., and Hasson, M. S. (2001) J. Bacteriol. 193, 680-686). Here we report two new crystal structure of the M. thermophila enzyme in the presence of substrate and transition state analogs. The enzyme co-crystallized with the ATP analog adenosine 5'-[gamma-thio]triphosphate contained AMP adjacent to thiopyrophosphate in the active site cleft of monomer B. The enzyme co-crystallized with ADP, acetate, Al(3+), and F(-) contained a linear array of ADP-AlF(3)-acetate in the active site cleft of monomer B. Together, the structures clarify the substrate binding sites and support a direct in-line transfer mechanism in which AlF(3) mimics the meta-phosphate transition state. Monomers A of both structures contained ADP and sulfate, and the active site clefts were closed less than in monomers B, suggesting that domain movement contributes to catalysis. The finding that His(180) was in close proximity to AlF(3) is consistent with a role for stabilization of the meta-phosphate that is in agreement with a previous report indicating that this residue is essential for catalysis. Residue Arg(241) was also found adjacent to AlF(3), consistent with a role for stabilization of the transition state. Kinetic analyses of Arg(241) and Arg(91) replacement variants indicated that these residues are essential for catalysis and also indicated a role in binding acetate.     

Kinetic and structural evidence for the importance of Tyr236 for the integrity of the Mo active site in a bacterial sulfite dehydrogenase

The sulfite dehydrogenase from Starkeya novella is the only known sulfite-oxidizing enzyme that forms a permanent heterodimeric complex between a molybdenum and a heme c-containing subunit and can be crystallized in an electron transfer competent conformation. Tyr236 is a highly conserved active site residue in sulfite oxidoreductases and has been shown to interact with a nearby arginine and a molybdenum-oxo ligand that is involved in catalysis. We have created a Tyr236 to Phe substitution in the SorAB sulfite dehydrogenase. The purified SDH(Y236F) protein has been characterized in terms of activity, structure, intramolecular electron transfer, and EPR properties. The substituted protein exhibited reduced turnover rates and substrate affinity as well as an altered reactivity toward molecular oxygen as an electron acceptor. Following reduction by sulfite and unlike SDH(WT), the substituted enzyme was reoxidized quickly in the presence of molecular oxygen, a process reminiscent of the reactions of the sulfite oxidases. SDH(Y236F) also exhibited the pH-dependent CW-EPR signals that are typically observed in vertebrate sulfite oxidases, allowing a direct link of CW-EPR properties to changes caused by a single-amino acid substitution. No quantifiable electron transfer was seen in laser flash photolysis experiments with SDH(Y236F). The crystal structure of SDH(Y236F) clearly shows that as a result of the substitution the hydrogen bonding network surrounding the active site is disturbed, resulting in an increased mobility of the nearby arginine. These disruptions underline the importance of Tyr236 for the integrity of the substrate binding site and the optimal alignment of Arg55, which appears to be necessary for efficient electron transfer.     

Rat mast cell carboxypeptidase: amino acid sequence and evidence of enzyme activity within mast cell granules

The amino acid sequence of rat mast cell carboxypeptidase has been determined. The major form has 308 residues; a minor form has an additional (glutamyl) residue at the amino terminus that may indicate an alternate cleavage site during zymogen activation. The enzyme is homologous to pancreatic carboxypeptidases A and B, with conservation of the functional amino acid residues of the active site. The putative substrate binding site resembles that of carboxypeptidase A, although other structural features bear more similarity to carboxypeptidase B. Mast cell carboxypeptidase retains enzymatic activity toward a peptide substrate (angiotensin I) while bound within the granular matrix of the rat connective tissue mast cells. Evidence is presented to suggest that a cluster of positively charged lysyl and arginyl residues binds the enzyme to the negatively charged heparin of the granular matrix but leaves the active site exposed to bind and cleave peptide substrates.