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Αn α-amylase (TVA II) from Thermoactinomyces vulgaris R-47 efficiently hydrolyzes α-1,4-glucosidic linkages of pullulan to produce panose in addition to hydrolyzing starch. TVA II also hydrolyzes α-1,4-glucosidic linkages of cyclodextrins and α-1,6-glucosidic linkages of isopanose. To clarify the basis for this wide substrate specificity of TVA II, we soaked 43-α-panosylpanose (43-P2) (a pullulan hydrolysate composed of two panosyl units) into crystals of D325N inactive mutated TVA II. We then determined the crystal structure of TVA II complexed with 42-α-panosylpanose (42-P2), which was produced by transglycosylation from 43-P2, at 2.2-Å resolution. The shape of the active cleft of TVA II is unique among those of α-amylase family enzymes due to a loop (residues 193–218) that is located at the end of the cleft around the nonreducing region and forms a ‘dam’-like bank. Because this loop is short in TVA II, the active cleft is wide and shallow around the nonreducing region. It is assumed that this short loop is one of the reasons for the wide substrate specificity of TVA II. While Trp356 is involved in the binding of Glc +2 of the substrate, it appears that Tyr374 in proximity to Trp356 plays two roles: one is fixing the orientation of Trp356 in the substrate-liganded state and the other is supplying the water that is necessary for substrate hydrolysis. [ABSTRACT FROM AUTHOR]

In order to determine the amino-terminal sequence requirements for protein N-myristoylation, site-directed mutagenesis of the N-terminal region was performed using tumor necrosis factor (TNF) mutants as model substrate proteins. Subsequently, the susceptibility of these mutants to protein N-myristoylation was evaluated by metabolic labeling in an in vitro translation system using rabbit reticulocyte lysate. A TNF mutant having the sequence MGAAAAAAAA at its N-terminus was used as the starting sequence to identify elements critical for protein N-myristoylation. Sequential vertical-scanning mutagenesis of amino acids at a distinct position in this model N-terminal sequence revealed the major sequence requirements for protein N-myristoylation: the combination of amino acids at position 3 and 6 constitutes a major determinant for the susceptibility to protein N-myristoylation. When Ser was located at position 6, 11 amino acids (Gly, Ala, Ser, Cys, Thr, Val, Asn, Leu, Ile, Gln, His) were permitted at position 3 to direct efficient protein N-myristoylation. In this case, the presence of Lys at position 7 was found to affect the amino acid requirement at position 3 and Lys became permitted at this position. When Ser was not located at position 6, only 3 amino acids (Ala, Asn, Gln) were permitted at position 3 to direct efficient protein N-myristoylation. The amino acid requirements found in this study were fully consistent with the N-terminal sequence of 78 N-myristoylated proteins in which N-myristoylation was experimentally verified. These observations strongly indicate that the combination of amino acids at position 3, 6 and 7 is a major determinant for protein N-myristoylation. [ABSTRACT FROM AUTHOR]

Through comparison with the high-resolution structure of Clostridium symbiosum glutamate dehydrogenase, the different substrate specificities of the homologous enzymes phenylalanine dehydrogenase and leucine dehydrogenase were attributed to two residues, glycine 124 and leucine 307, in Bacillus sphaericus phenylalanine dehydrogenase, which are replaced with alanine and valine in leucine dehydrogenases [Britton, K.L., Baker, P.J., Engel, P.C., Rice, D.W. & Stillman, T.J. (1993) J. Mol. Biol. 234, 938–945]. As predicted, making these substitutions in phenylalanine dehydrogenase decreased the specific activity towards aromatic substrates and enhanced the activity towards some aliphatic amino acids in standard assays with fixed concentrations of both substrates [Seah, S.Y.K., Britton, K.L., Baker, P.J., Rice, D.W., Asano, Y. & Engel, P.C. (1995) FEBS Lett. 370, 93–96]. This study did not, however, distinguish effects on affinity from those on maximum catalytic rate. A fuller kinetic characterization of the single- and double-mutant enzymes now reveals that the extent of the shift in specificity was underestimated in the earlier study. The maximum catalytic rates for aromatic substrates are reduced for all the mutants, but, in addition, the apparent Km values are higher for the single-mutant G124A and double-mutant G124A/L307V compared with the wild-type enzyme. Conversely, specificity constants ( kcat/ Km) for the nonpolar aliphatic amino acids and the corresponding 2-oxoacids for the mutants are all markedly higher than for the wild type, with up to a 40-fold increase for l-norvaline and a 100-fold increase for its 2-oxoacid in the double mutant. In some cases a favourable change in Km was found to outweigh a smaller negative change in kcat. These results emphasize the risk of misjudging the outcome of protein engineering experiments through too superficial an analysis. Overall, however, the success of the predictions from molecular modelling indicates the usefulness of this strategy for engineering new specificities, even in advance of more detailed 3D structural information. [ABSTRACT FROM AUTHOR]

Although there is a significant knowledge about mammalian metallocarboxypeptidases, the data available on this family of enzymes is very poor for invertebrate forms. Here we present the biochemical characterization of a metallocarboxypeptidase from the insect Helicoverpa armigera (Lepidoptera: Noctuidae), a devastating pest spread in subtropical regions of Europe, Asia, Africa and Oceania. The zymogen of this carboxypeptidase (PCPAHa) has been expressed at high levels in a Pichia pastoris system and shown to display the characteristics of the enzyme purified from the insect midgut. The in vitro activation process of the proenzyme differs significantly from the mammalian ones. The lysine-specific endoprotease LysC activates PCPAHa four times more efficiently than trypsin, the general activating enzyme for all previously studied metalloprocarboxypeptidases. LysC and trypsin independently use two different activation targets and the presence of sugars in the vicinity of the LysC activation point affects the activation process, indicating a possible modulation of the activation mechanism. During the activation with LysC the prodomain is degraded, while the carboxypeptidase moiety remains intact except for a C-terminal octapeptide that is rapidly released. Interestingly, the sequence at the cleavage point for the release of the octapeptide is also found at the boundary between the activation peptide and the enzyme moieties. The active enzyme (CPAHa) is shown to have a very broad substrate specificity, as it appears to be the only known metallocarboxypeptidase capable of efficiently hydrolysing basic and aliphatic residues and, to a much lower extent, acidic residues. Two carboxypeptidase inhibitors, from potato and leech, were tested against CPAHa. The former, of vegetal origin, is the most efficient metallocarboxypeptidase inhibitor described so far, with a K i in the pm range. [ABSTRACT FROM AUTHOR]

The thiamin diphosphate-dependent enzyme indolepyruvate decarboxylase catalyses the formation of indoleacetaldehyde from indolepyruvate, one step in the indolepyruvate pathway of biosynthesis of the plant hormone indole-3-acetic acid. The crystal structure of this enzyme from Enterobacter cloacae has been determined at 2.65 Å resolution and refined to a crystallographic R-factor of 20.5% (R free 23.6%). The subunit of indolepyruvate decarboxylase contains three domains of open α/β topology, which are similar in structure to that of pyruvate decarboxylase. The tetramer has pseudo 222 symmetry and can be described as a dimer of dimers. It resembles the tetramer of pyruvate decarboxylase from Zymomonas mobilis , but with a relative difference of 20° in the angle between the two dimers. Active site residues are highly conserved in indolepyruvate/pyruvate decarboxylase, suggesting that the interactions with the cofactor thiamin diphosphate and the catalytic mechanisms are very similar. The substrate binding site in indolepyruvate decarboxylase contains a large hydrophobic pocket which can accommodate the bulky indole moiety of the substrate. In pyruvate decarboxylases this pocket is smaller in size and allows discrimination of larger vs. smaller substrates. In most pyruvate decarboxylases, restriction of cavity size is due to replacement of residues at three positions by large, hydrophobic amino acids such as tyrosine or tryptophan. [ABSTRACT FROM AUTHOR]

Enterobacter cloacae , isolated from the rhizosphere of cucumbers, produces large amounts of indole-3-acetic acid. Indolepyruvate decarboxylase, the key enzyme in the biosynthetic pathway of indole-3-acetic acid, catalyses the formation of indole-3-acetaldehyde and carbon dioxide from indole-3-pyruvic acid. The enzyme requires the cofactors thiamine diphosphate and magnesium ions for catalytic activity. Recombinant indolepyruvate decarboxylase was purified from the host Escherichia coli strain JM109. Specificity of the enzyme for the substrates indole-3-pyruvic acid, pyruvic acid, benzoylformic acid, and seven benzoylformic acid analogues was investigated using a continuous optical assay. Stopped-flow kinetic data showed no indication for substrate activation in the decarboxylation reaction of indole-3-pyruvic acid, pyruvic acid or benzoylformic acid. Size exclusion chromatography and small angle X-ray solution scattering experiments suggested the tetramer as the catalytically active state and a pH-dependent subunit association equilibrium. Analysis of the kinetic constants of the benzoylformic acid analogues according to Hansch et al . [Hansch, C., Leo, A., Unger, S.H., Kim, K.H., Nikaitani, D & Lien, E.J. (1973) J. Med. Chem. 16, 1207–1216] and comparison with indole-3-pyruvic acid conversion by pyruvate decarboxylases from Saccharomyces cerevisiae and Zymomonas mobilis provided some insight into the catalytic mechanism of indolepyruvate decarboxylase. [ABSTRACT FROM AUTHOR]

NMR spectroscopy and X-ray crystallography have provided important insight into structural features of phenylalanine hydroxylase (PAH) and tyrosine hydroxylase (TH). Nevertheless, significant problems such as the substrate specificity of PAH and the different susceptibility of TH to feedback inhibition by l-3,4-dihydroxyphenylalanine (l-DOPA) compared with dopamine (DA) remain unresolved. Based on the crystal structures 5pah for PAH and 2toh for TH (Protein Data Bank), we have used molecular docking to model the binding of 6(R )-l-erythro-5,6,7,8-tetrahydrobiopterin (BH4 ) and the substrates phenylalanine and tyrosine to the catalytic domains of PAH and TH. The amino acid substrates were placed in positions common to both enzymes. The productive position of tyrosine in TH·BH4 was stabilized by a hydrogen bond with BH4 . Despite favorable energy scores, tyrosine in a position trans to PAH residue His290 or TH residue His336 interferes with the access of the essential cofactor dioxygen to the catalytic center, thereby blocking the enzymatic reaction. DA and l-DOPA were directly coordinated to the active site iron via the hydroxyl residues of their catechol groups. Two alternative conformations, rotated 180° around an imaginary iron–catecholamine axis, were found for DA and l-DOPA in PAH and for DA in TH. Electrostatic forces play a key role in hindering the bidentate binding of the immediate reaction product l-DOPA to TH, thereby saving the enzyme from direct feedback inhibition. [ABSTRACT FROM AUTHOR]

Although the α-chymases of primates and dogs are known as chymotrypsin-like proteases, the enzymatic properties of rodent α-chymases (rat mast cell protease 5/rMCP-5 and mouse mast cell protease 5/mMCP-5) have not been fully understood. We report that recombinant rMCP-5 and mMCP-5 are elastase-like proteases, not chymotrypsin-like proteases. An enzyme assay using chromogenic peptidyl substrates showed that mast cell protease-5s (MCP-5s) have a clear preference for small aliphatic amino acids (e.g. alanine, isoleucine, valine) in the P1 site of substrates. We used site-directed mutagenesis and computer modeling approaches to define the determinant residue for the substrate specificity of mMCP-5, and found that the mutant possessing a Gly substitution of the Val at position 216 (V216G) lost elastase-like activity but acquired chymase activity, suggesting that the Val216 dominantly restricts the substrate specificity of mMCP-5. Structural models of mMCP-5 and the V216G mutant based on the crystal structures of serine proteases (rMCP-2, human cathepsin G, and human chymase) revealed the active site differences that can account for the marked differences in substrate specificity of the two enzymes between elastase and chymase. These findings suggest that rodent α-chymases have unique biological activity different from the chymases of other species. [ABSTRACT FROM AUTHOR]

The HIV-1 proteinase (PR) has proved to be a good target for antiretroviral therapy of AIDS, and various PR inhibitors are now in clinical use. However, there is a rapid selection of viral variants bearing mutations in the proteinase that are resistant to clinical inhibitors. Drug resistance also involves mutations of the nucleocapsid/p1 and p1/p6 cleavage sites of Gag, both in vitro and in vivo . Cleavages at these sites have been shown to be rate limiting steps for polyprotein processing and viral maturation. Furthermore, these sites show significant sequence polymorphism, which also may have an impact on virion infectivity. We have studied the hydrolysis of oligopeptides representing these cleavage sites with representative mutations found as natural variations or that arise as resistant mutations. Wild-type and five drug resistant PRs with mutations within or outside the substrate binding site were tested. While the natural variations showed either increased or decreased susceptibility of peptides toward the proteinases, the resistant mutations always had a beneficial effect on catalytic efficiency. Comparison of the specificity changes obtained for the various substrates suggested that the maximization of the van der Waals contacts between substrate and PR is the major determinant of specificity: the same effect is crucial for inhibitor potency. The natural nucleocapsid/p1 and p1/p6 sites do not appear to be optimized for rapid hydrolysis. Hence, mutation of these rate limiting cleavage sites can partly compensate for the reduced catalytic activity of drug resistant mutant HIV-1 proteinases. [ABSTRACT FROM AUTHOR]

Penicillin acylase of Escherichia coli catalyses the hydrolysis and synthesis of β-lactam antibiotics. To study the role of hydrophobic residues in these reactions, we have mutated three active-site phenylalanines. Mutation of αF146, βF24 and βF57 to Tyr, Trp, Ala or Leu yielded mutants that were still capable of hydrolysing the chromogenic substrate 2-nitro-5-[(phenylacetyl)amino]-benzoic acid. Mutations on positions αF146 and βF24 influenced both the hydrolytic and acyl transfer activity. This caused changes in the transferase/hydrolase ratios, ranging from a 40-fold decrease for αF146Y and αF146W to a threefold increase for αF146L and βF24A, using 6-aminopenicillanic acid as the nucleophile. Further analysis of the βF24A mutant showed that it had specificity constants (k cat /K m ) for p -hydroxyphenylglycine methyl ester and phenylglycine methyl ester that were similar to the wild-type values, whereas the specificity constants for p -hydroxyphenylglycine amide and phenylglycine amide had decreased 10-fold, due to a decreased k cat value. A low amidase activity was also observed for the semisynthetic penicillins amoxicillin and ampicillin and the cephalosporins cefadroxil and cephalexin, for which the k cat values were fivefold to 10-fold lower than the wild-type values. The reduced specificity for the product and the high initial transferase/hydrolase ratio of βF24A resulted in high yields in acyl transfer reactions. [ABSTRACT FROM AUTHOR]

SulA protein is known to be one of the physiological substrates of Lon protease, an ATP-dependent protease from Escherichia coli . In this study, we investigated the cleavage specificity of Lon protease toward SulA protein. The enzyme was shown to cleave ≈ 27 peptide bonds in the presence of ATP. Among them, six peptide bonds were cleaved preferentially in the early stage of digestion, which represented an apparently unique cleavage sites with mainly Leu and Ser residues at the P1 , and P1 ′ positions, respectively, and one or two Gln residues in positions P2 –P5 . They were located in the central region and partly in the C-terminal region, both of which are known to be important for the function of SulA, such as inhibition of cell growth and interaction with Lon protease, respectively. The other cleavage sites did not represent such consensus sequences, though hydrophobic or noncharged residues appeared to be relatively preferred at the P1 sites. On the other hand, the cleavage in the absence of ATP was very much slower, especially in the central region, than in the presence of ATP. The central region was predicted to be rich in α helix and β sheet structures, suggesting that the enzyme required ATP for disrupting such structures prior to cleavage. Taken together, SulA is thought to contain such unique cleavage sites in its functionally and structurally important regions whose preferential cleavage accelerates the ATP-dependent degradation of the protein by Lon protease. [ABSTRACT FROM AUTHOR]

The nitrilase AtNIT1 from Arabidopsis thaliana was overexpressed in Escherichia coli with an N-terminal His6 tag and purified by zinc chelate affinity chromatography in a single step almost to homogeneity in a 68% yield with a specific activity of 34.1 U·mg-1 . The native enzyme (≈ 450 kDa) consists of 11–13 subunits (38 kDa). The temperature optimum was determined to be 35 °C, and a pH optimum of 9 was found. Thus, recombinant AtNIT1 resembles in its properties the native enzyme and the nitrilase from Brassica napus . The stability of AtNIT1 could be significantly improved by the addition of dithiothreitol and EDTA. The substrate range of AtNIT1 differs considerably from those of bacterial nitrilases. Aliphatic nitriles are the most effective substrates, showing increasing rates of hydrolysis with increasing size of the residues, as demonstrated in the series butyronitrile, octanenitrile, phenylpropionitrile. In comparison with 3-indolylacetonitrile, the rate of hydrolysis of 3-phenylpropionitrile is increased by a factor of 330, and the K m value is reduced by a factor of 23. With the exception of fluoro, substituents in the α position to the nitrile function completely inhibit the hydrolysis. [ABSTRACT FROM AUTHOR]

Enzymatic properties of barley α-amylase 1 (AMY1) are altered as a result of amino acid substitutions at subsites -5/-6 (Cys95→Ala/Thr) and +1/+2 (Met298→Ala/Asn/Ser) as well as in the double mutants, Cys95→Ala/Met298→Ala/Asn/Ser. Cys95→Ala shows 176% activity towards insoluble Blue Starch compared to wild-type AMY1, kcat of 142 and 211% towards amylose DP17 and 2-chloro-4-nitrophenyl β-d-maltoheptaoside (Cl-PNPG7), respectively, but fivefold to 20-fold higher Km. The Cys95→Thr-AMY1 AMY2 isozyme mimic exhibits the intermediary behaviour of Cys95→Ala and wild-type. Met298→Ala/Asn/Ser have slightly higher to slightly lower activity for starch and amylose, whereas kcat and kcat/Km for Cl-PNPG7 are ≤ 30% and ≤ 10% of wild-type, respectively. The activity of Cys95→Ala/Met298→Ala/Asn/Ser is 100–180% towards starch, and the kcat/Km is 15–30%, and 0.4–1.1% towards amylose and Cl-PNPG7, respectively, emphasizing the strong impact of the Cys95→Ala mutation on activity. The mutants therefore prefer the longer substrates and the specificity ratios of starch/Cl-PNPG7 and amylose/Cl-PNPG7 are 2.8- to 270-fold and 1.2- to 60-fold larger, respectively, than of wild-type. Bond cleavage analyses show that Cys95 and Met298 mutations weaken malto-oligosaccharide binding near subsites -5 and +2, respectively. In the crystal structure Met298 CE and SD (i.e., the side chain methyl group and sulfur atom) are near C(6) and O(6) of the rings of the inhibitor acarbose at subsites +1 and +2, respectively, and Met298 mutants prefer amylose for glycogen, which is hydrolysed with a slightly lower activity than by wild-type. Met298 AMY1 mutants and wild-type release glucose from the nonreducing end of the main-chain of 6′′′-maltotriosyl-maltohexaose thus covering subsites -1 to +5, while productive binding of unbranched substrate involves subsites -3 to +3. [ABSTRACT FROM AUTHOR]

The Trypanosoma cruzi cysteine protease cruzipain contains a 130-amino-acid C-terminal extension, in addition to the catalytic domain. Natural cruzipain is a complex of isoforms, because of the simultaneous expression of several genes, and the presence of either high mannose-type, hybrid monoantennary-type or complex biantenary-type oligosacharide chains at Asn255 of the C-terminal extension. Cruzipain and its recombinant form without this extension (cruzain) were studied comparatively in this work. S2 to S2′ subsite specificities of these enzymes were examined using four series of substrates derived from the internally quenched fluorescent peptide Abz-KLRFSKQ-EDDnp (Abz, ortho-aminobenzoic acid; EDDnp, N-(2,4-dinitrophenyl)-ethylenediamine). Large differences in the kinetic parameters were not observed between the enzymes; however, Km values were consistently lower for the hydrolysis of most of the substrates by cruzain. No difference in the pH–activity profile between the two enzymes was found, but in 1 m NaCl cruzipain presented a kcat value significantly higher than that of cruzain. The activation energy of denaturation for the enzymes did not differ significantly; however, a negative entropy value was observed for cruzipain denaturation whereas the value for cruzain was positive. We determined the individual rate constants (k1, substrate diffusion; k-1, substrate dissociation; k2, acylation; k3, deacylation) and the respective activation energies and entropies for hydrolysis of Abz-KLRFSKQ-EDDnp determining the temperature dependence of the Michaelis–Menten parameters kcat/Km and kcat as previously described [Ayala, Y.M. & Di Cera, E. (2000) Protein Sci. 9, 1589–1593]. Differences between the two enzymes were clearly detected in the activation energies E1 and E-1, which are significantly higher for cruzipain. The corresponding ΔS1 and ΔS-1 were positive and significantly higher for cruzipain than for cruzain. These results indicate the presence of a larger energy barrier for cruzipain relating to substrate diffusion and dissociation, which could be related to the C-terminal extension and/or glycosylation state of cruzipain. [ABSTRACT FROM AUTHOR]

In earlier attempts to shift the substrate specificity of glutamate dehydrogenase (GDH) in favour of monocarboxylic amino-acid substrates, the active-site residues K89 and S380 were replaced by leucine and valine, respectively, which occupy corresponding positions in leucine dehydrogenase. In the GDH framework, however, the mutation S380V caused a steric clash. To avoid this, S380 has been replaced with alanine instead. The single mutant S380A and the combined double mutant K89L/S380A were satisfactorily overexpressed in soluble form and folded correctly as hexameric enzymes. Both were purified successfully by Remazol Red dye chromatography as routinely used for wild-type GDH. The S380A mutant shows much lower activity than wild-type GDH with glutamate. Activities towards monocarboxylic substrates were only marginally altered, and the pH profile of substrate specificity was not markedly altered. In the double mutant K89L/S380A, activity towards glutamate was undetectable. Activity towards l-methionine, l-norleucine and l-norvaline, however, was measurable at pH 7.0, 8.0 and 9.0, as for wild-type GDH. Ala163 is one of the residues that lines the binding pocket for the side chain of the amino-acid substrate. To explore its importance, the three mutants A163G, K89L/A163G and K89L/S380A/A163G were constructed. All three were abundantly overexpressed and showed chromatographic behaviour identical with that of wild-type GDH. With A163G, glutamate activity was lower at pH 7.0 and 8.0, but by contrast higher at pH 9.0 than with wild-type GDH. Activities towards five aliphatic amino acids were remarkably higher than those for the wild-type enzyme at pH 8.0 and 9.0. In addition, the mutant A163G used l-aspartate and l-leucine as substrates, neither of which gave any detectable activity with wild-type GDH. Compared with wild-type GDH, the A163 mutant showed lower catalytic efficiencies and higher Km values for glutamate/2-oxoglutarate at pH 7.0, but a similar kcat/Km value and lower Km at pH 8.0, and a nearly 22-fold lower S0.5 (substrate concentration giving half-saturation under conditions where Michaelis–Menten kinetics does not apply) at pH 9.0. Coupling the A163G mutation with the K89L mutation markedly enhanced activity (100–1000-fold) over that of the single mutant K89L towards monocarboxylic amino acids, especially l-norleucine and l-methionine. The triple mutant K89L/S380A/A163G retained a level of activity towards monocarboxylic amino acids similar to that of the double mutant K89L/A163G, but could no longer use glutamate as substrate. In terms of natural amino-acid substrates, the triple mutant represents effective conversion of a glutamate dehydrogenase into a methionine dehydrogenase. Kinetic parameters for the reductive amination reaction are also reported. At pH 7 the triple mutant and K89L/A163G show 5 to 10-fold increased catalytic efficiency, compared with K89L, towards the novel substrates. In the oxidative deamination reaction, it is not possible to estimate kcat and Km separately, but for reductive amination the additional mutations have no significant effect on kcat at pH 7, and the increase in catalytic efficiency is entirely attributable to the measured decrease in Km. At pH 8 the enhancement of catalytic efficiency with the novel substrates was much more striking (e.g. for norleucine ≈ 2000-fold compared with wild-type or the K89L mutant), but it was not established whether this is also exclusively due to more favourable Michaelis constants. [ABSTRACT FROM AUTHOR]

The hydrophobic S1′ subsite is one of the major determinants of the substrate specificity of thermolysin and related M4 family proteases. In the thermolysin-like protease (TLP) produced by Bacillus stearothermophilus (TLP-ste), the hydrophobic S1′ subsite is mainly formed by Phe130, Phe133, Val139 and Leu202. In the present study, we have examined the effects of replacing Leu202 by smaller (Gly, Ala, Val) and larger (Phe, Tyr) hydrophobic residues. The mutational effects showed that the wild-type S1′ pocket is optimal for binding leucine side chains. Reduction of the size of residue 202 resulted in a higher efficiency towards substrates with Phe in the P1′ position. Rather unexpectedly, the Leu202→Phe and Leu202→Tyr mutations, which were expected to decrease the size of the S1′ subsite, resulted in a large increase in activity towards dipeptide substrates with Phe in the P1′ position. This is probably due to the fact that 202Phe and 202Tyr adopt a second possible rotamer that opens up the subsite compared to Leu202, and also favours interactions with the substrate. To validate these results, we constructed variants of thermolysin with changes in the S1′ subsite. Thermolysin and TLP-ste variants with identical S1′ subsites were highly similar in terms of their preference for Phe vs. Leu in the P1′ position. [ABSTRACT FROM AUTHOR]

The physiological function of the stilbene synthase (STS) from groundnut (Arachis hypogaea) is the formation of resveratrol. The enzyme uses 4-coumaroyl-CoA, performs three condensations with malonyl-CoA, and folds the resulting tetraketide into a new aromatic ring system. We investigated the capacity for building novel and unusual polyketides from alternative substrates. Three types of products were obtained: (a) complete reaction (stilbene-type), (b) three condensations without formation of an aromatic ring (CTAL-type pyrone derailment), and (c) two condensations (BNY-type pyrone derailment). All product types were obtained from 4-fluorocinnamoyl-CoA and analogs in which the coumaroyl moiety was replaced by furan or thiophene. Only type (b) and (c) products were synthesized from other 4-substituted 4-coumaroyl-CoA analogs (-Cl, -Br, -OCH3). Benzoyl-CoA, phenylacetyl-CoA, and medium chain aliphatic CoA esters were poor substrates, and the majority of the products were of type (c). The results show that minor modifications can be used to direct the enzyme reaction to form a variety of different and new products. Manipulation of the biosynthesis of polyketides by synthetic analogs could lead to the development of a chemical library of pharmaceutically interesting novel polyketides. [ABSTRACT FROM AUTHOR]

Human T-cell leukemia virus type-1 (HTLV-1) is associated with a number of human diseases. Based on the therapeutic success of human immunodeficiency virus type 1 (HIV-1) PR inhibitors, the proteinase (PR) of HTLV-1 is a potential target for chemotherapy. To facilitate the design of potent inhibitors, the subsite specificity of HTLV-1 PR was characterized and compared to that of HIV-1 PR. Two sets of substrates were used that contained single amino-acid substitutions in peptides representing naturally occurring cleavage sites in HIV-1 and HTLV-1. The original HIV-1 matrix/capsid cleavage site substrate and most of its substituted peptides were not hydrolyzed by the HTLV-1 enzyme, except for those with hydrophobic residues at the P4 and P2 positions. On the other hand, most of the peptides representing the HTLV-1 capsid/nucleocapsid cleavage site were substrates of both enzymes. A large difference in the specificity of HTLV-1 and HIV-1 proteinases was demonstrated by kinetic measurements, particularly with regard to the S4 and S2 subsites, whereas the S1 subsite appeared to be more conserved. A molecular model of the HTLV-1 PR in complex with this substrate was built, based on the crystal structure of the S9 mutant of Rous sarcoma virus PR, in order to understand the molecular basis of the enzyme specificity. Based on the kinetics of shortened analogs of the HTLV-1 substrate and on analysis of the modeled complex of HTLV-1 PR with substrate, the substrate binding site of the HTLV-1 PR appeared to be more extended than that of HIV-1 PR. Kinetic results also suggested that the cleavage site between the capsid and nucleocapsid protein of HTLV-1 is evolutionarily optimized for rapid hydrolysis. [ABSTRACT FROM AUTHOR]

Nitrilase-containing resting cells of Rhodococcus rhodochrous J1 converted acrylonitrile and benzonitrile to the corresponding acids, but the purified nitrilase hydrolyzed only benzonitrile, and not acrylonitrile. The activity of the purified enzyme towards acrylonitrile was recovered by preincubation with 10 mm benzonitrile, but not by preincubation with aliphatic nitriles such as acrylonitrile. It was shown by light-scattering experiments, that preincubation with benzonitrile led to the assembly of the inactive, purified and homodimeric 80-kDa enzyme to its active 410-kDa aggregate, which was proposed to be a decamer. Furthermore, the association concomitant with the activation was reached after dialysis of the enzyme against various salts and organic solvents, with the highest recovery reached at 10% saturated ammonium sulfate and 50% (v/v) glycerol, and by preincubation at increased temperatures or enzyme concentrations. [ABSTRACT FROM AUTHOR]

The availability of a high-resolution structure of the Thermobifida fusca endocellulase Cel6A catalytic domain makes this enzyme ideal for structure-based efforts to engineer cellulases with high activity on native cellulose. In order to determine the role of conserved, noncatalytic residues in cellulose hydrolysis, 14 mutations of six conserved residues in or near the Cel6A active-site cleft were studied for their effects on catalytic activity, substrate specificity, processivity and ligand-binding affinity. Eleven mutations were generated by site-directed mutagenesis using PCR, while three were from previous studies. All the CD spectra of the mutant enzymes were indistinguishable from that of Cel6A indicating that the mutations did not dramatically change protein conformation. Seven mutations in four residues (H159, R237, K259 and E263) increased activity on carboxymethyl cellulose (CM-cellulose), with K259H (in glucosyl subsite -2) creating the highest activity (370%). Interestingly, the other mutations in these residues reduced CM-cellulose activity. Only the K259H enzyme retained more activity on acid-swollen cellulose than on filter paper, suggesting that this mutation affected the rate-limiting step in crystalline cellulose hydrolysis. All the mutations lowered activity on cellotriose and cellotetraose, but two mutations, both in subsite +1 (H159S and N190A), had higher kcat/Km values (6.6-fold and 5.0-fold, respectively) than Cel6A on 2,4-dinitrophenyl-β-d-cellobioside. Measurement of enzyme : ligand dissociation constants for three methylumbelliferyl oligosaccharides and cellotriose showed that all mutant enzymes bound these ligands either to the same extent as or more weakly than Cel6A. These results show that conserved noncatalytic residues can profoundly affect Cel6A activity and specificity. [ABSTRACT FROM AUTHOR]

The acceptor specificities of rat liver Gal(β1-4)GlcNAc α-2,6-sialytransferase, recombinant full-length human liver Gal(β1-4)GlcNAc α-2,6-sialytransferase, and a soluble form of recombinant rat liver Gal(β1-3/4) GlcNAc α-2,6-sialytransferase were studied with a panel of analogues of the trisaccharide Gal(β1-4)GlcNAc(β1-2)Man(α1-O)(CH2)7CH3. These analogues contain structural variants of D-galactose, modified at either C3, C4 or C5 by deoxygenation, fluorination, O-methylation, epimerization, or by the introduction of an amino group. In addition, the enantiomer of D-galactose is included. The α-2,6-sialytransferases tolerated most of the modifications at the galactose residue to some extent, whereas the α-2,3-sialytransferases displayed a narrower specifity. Molecular dynamics simultations were performed in order to correlate enzymatic activity to three-dimensional structure. Ineffective acceptors for rat liver α-2,6-sialytransferase were shown to be inhibitory towards the enzyme; likewise, the α-2,6-sialytransferase was found to be inhibited by all non-substrates. Modified sialyloligosaccharides was found to be inhibited by all non-substrates. Modified sialyloligosaccharides were obtained on a milligram scale by incubation of effective acceptors with one of the three enzymes, and characterized by 500-MHz 1H-NMR spectroscopy. [ABSTRACT FROM AUTHOR]

Porcine pancreatic phospholipase A2 (PLA2) was studied by site-directed mutagenesis. Arg53 and/or Lys56 were replaced by a methionine (R53M or K56M, respectively) in combination with the Tyr69→Phe (Y69F) substitution. These substitutions improved the activity on micellar and monomeric zwitterionic substrates and reduced the activity on negatively charged substrates compared to the Y69F mutant. With the neutral substrate 1,2-didodecanoyl-sn-glycerol-3-dimethyl phosphate (Lau2GroMe2P) a 20-fold increase of activity was observed for the 69F53M56M mutant, whereas this mutant showed a lower activity than native PLA2 on zwitterionic substrates. Thus the ratio Lau2GroMe2P/Lau2GroPCho has become 65 times higher for 69F53M56M compared to native phospholipase A2, illustrating that the substrate specificity has changed enormously. The methionine substitutions were also prepared in a 69F mutant in which a part of the surface loop (residues 62–66) was deleted. Also in this deletion mutant these substitutions showed a similar effect as the substitutions in the native 69F mutant. Furthermore it was shown that deletion of the loop increases the activity on micellar lecithins and negatively charged micellar substrates, but reduces the activity on Lau2GroMe2P. Therefore it can be concluded that the loop is important for the recognition of substrates. We also show that the loop plays a role in the dimerization of these proteins. Dimerization may account for the high activities observed for some mutants acting on monomeric substrate. [ABSTRACT FROM AUTHOR]

Bacillus circulans F-2 amylase-pullulanase enzyme (APE) displayed dual activity with respect to glycosidic bond cleavage. The enzyme was active on α-1,6 bonds in pullulan, amylopectin, and glycogen, while it showed α-1,4 activity against malto-oligosaccharides, amylose, amylopectin, and soluble starch, but not pullulan. Kinetic analysis of the purified enzyme in a system which contained both pullulan and amylose as two competing substrates was used to distinguish the dual specificity of the enzyme from the single-substrate specificity known for pullulanases and α-amylases. Enzyme activities were inhibited by some metal ions, and by metal-chelating agents with a different mode. The enzyme-inhibitory results of amylase and pullulanase with Hg2+ and Co2+ ions were different, indicating that the activation mechanisms of both enzyme activities are different. Cyclomaltoheptaose inhibited both a-amylase and pullulanase activities with inhibition constants (Ki) of 0.029 and 0.06 mg/ml, respectively. Modification with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide confirmed a carboxy group at the active sites of both enzymes. The N-terminal sequence of the enzyme was: Ala-Asp-Ala-Lys-Lys-Thr-Pro-Gln-Gln-Gln-Phe-Asp-Ala-Leu-Trp-Ala-Ala-Gly-ILe-Val-Thr-Gly-Thr-Pro-Asp-Gly-Phe. The purified enzyme displayed Michaelis constant (Km) values of 0.55 mg/ml for amylose, and 0.71 mg/ml for pullulan. When both amylose and pullulan were simultaneously present, the observed rate of product formation closely fitted a kinetic model in which the two substrates are hydrolyzed at different active sites. These results suggest that amylopullulanases, which possess both α-1,6 and α-1,4 cleavage activities at the same active site, should be distinguished from APEs, which contain both activities at different active sites on the same polypeptide. Also, it is proposed that the Enzyme Commission use the term ‘amylase-pullulanase enzyme’ to refer to enzymes which act on starch and cleave both α-1,6-bonds in pullulan and α-1,4 bonds in amylose at different active sites. [ABSTRACT FROM AUTHOR]

Very-long-chain acyl-coenzyme A dehydrogenase (VLCAD) is one of four enzymes which catalyze the initial step of the mitochondrial β-oxidation with different but overlapping substrate-chain-length specificities. A450P, a variant of VLCAD identified in a patient with VLCAD deficiency, showed abnormal substrate-chain-length specificity. Based on this mutation, we studied the relationship between the structure and substrate-chain-length specificity of VLCAD. When VLCAD was treated with trypsin, a homodimer protein of a 48-kDa polypeptide deprived of both the amino-terminal 22 amino acids and the carboxyl-terminal 145 amino acids of VLCAD was obtained. Six Ala450 variants and tryptic-VLCAD exhibited similar substrate specificities. Effects of long-chain acyl-CoA on the tryptic cleavage and changes in the catalytic properties by deprivation of the carboxyl-terminal region suggest that this region interacts with the fatty acyl moiety of long-chain acyl-CoA. Thus, both Ala450 and the carboxyl-terminal region, which are not shared by other acyl-CoA dehydrogenases, are likely to be the determinating factors in the substrate-chain-length specificity of VLCAD. [ABSTRACT FROM AUTHOR]

Duodenase, a serine protease from bovine duodenum mucosa, was located in endoplasmic reticulum, the Golgi secretory granules of epithelial cells and ducts of Brunner's glands by the A-gold immunocytochemical method. Duodenase exhibits trypsin-like and chymotrypsin-like specificities with a preference for substrates having lysine at the P1 and proline at the P2 positions. The kinetic constants for the hydrolysis of 21 potential duodenase substrates are reported. The best substrates were found to be αN-tosyiglycylprolyllysine 4-nitroanilide (kcat/Km of 35 000 M-1 s-1), α-N-succinylthreonytprotyllysine 4nitroanilide (kcat/Km of 18 000 M-1 s-1) and α-N-serylprolyllysine 4-nitroanilide (kcat/Km of 2600 M-1 s-1), all of which contain the P1-P3 sequence of the enteropeptidase zymogen/activation site. On the basis of its catalytic properties and sites of localization, duodenase has been postulated to be an activator of the enteropeptidase precursor. A tetradecapeptide (LVTQEVSPKIVGGS) having the P9-P5'sequence of the cleavage site of zymogen activation of bovine proenteropeptidase was synthesized, and kinetic parameters of its hydrolysis by duodenase were determined (Km of 87 µM; kcat of 1.4 s-1; kcat/Km of 16 000 M-1 s-1). Crystals of duodenase frozen in a stream of liquid nitrogen diffracted synchrotron Xrays to 0.2-nm resolution. [ABSTRACT FROM AUTHOR]

Degradation of protein antigens by cellular proteases is a crucial step in the initiation of a T-cell-mediated immune response. But still little is known about the enzymes responsible for the processing of antigens, including their specificity. In this paper, we show that the combination of automated N-terminal sequencing with a newly developed method for C-terminal sequencing of peptide pools generated by the aspartic proteases cathepsins D and E is a fast and/easy method to obtain detailed information of the substrate specificity of these endopeptidases. Using a 15-residue synthetic peptide library and a native protein as substrates, we confirm and extend the knowledge about the cleavage motif of cathepsin E where positions P1 and P1′ of the substrate must be occupied exclusively by hydrophobic amino acids with aromatic or aliphatic side chains. However, Val and Ile residues are not allowed m position P1. Position P2" accepts a broad range of amino acids, including charged and polar ones. Additional requirements concerning the substrate positions P3′ and P4′ were also defined by pool sequencing. Furthermore. pool sequencing analysis of melittin digests with the aspartic proteases cathepsin D and E provided evidence that both enzymes share the same cleavage motif, identical to the one derived from the peptide library, and the native protein. Therefore, pool sequencing analysis is a valuable and fast tool to determine the substrate specificity of any endopeptidase. [ABSTRACT FROM AUTHOR]

Ribonuclease P (RNase P) from wheat nuclei has been purified over 1000-fold, using wheat germ extract as starting material and a combination of poly(ethytenglycol) precipitation and column chromatograpliy. The enzyme was shown to be of nuclear origin by its characteristic ionic requirements: for optimum activity it requires 0.5-1.5 mM Mg2+, which can be partly replaced by Mn2+. With about 100 kDa, wheat nuclear RNase P has the lowest molecular mass reported so far for a eukaryotic RNase P. The enzyme has an isoelectric point of 5.0 and a buoyant density of 1.34 g/ml in CsCl, suggesting the presence of a nucleic acid component: it is, however, insensitive against treatment with micrococcal nuclease. Wheat germ RNase P requires an intact tertiary structure of the pre-tRNA substrate; its cleavage efficiency is also influenced by the presence of an intron, and by the nature of the 3' terminus of the substrate. The apparent Km and Vmax, for an intronless plant pre-tRNATyr are 10.3 nM and 1.12 fmol/min, respectively. [ABSTRACT FROM AUTHOR]

Porcine pancreatic phospholipase A2 (PLA2) was studied by site-directed mutagenesis. Arg53 and/or Lys56 were replaced by a methionine (R53M or K56M, respectively) in combination with the Tyr69→Phe (Y69F) substitution. These substitutions improved the activity on micellar and monomeric zwitterionic substrates and reduced the activity on negatively charged substrates compared to the Y69F mutant. With the neutral substrate 1,2-didodecanoyl-sn-glycerol-3-dimethyl phosphate (Lau2GroMe2P) a 20-fold increase of activity was observed for the 69F53M56M mutant, whereas this mutant showed a lower activity than native PLA2 on zwitterionic substrates. Thus the ratio Lau2GroMe2P/Lau2GroPCho has become 65 times higher for 69F53M56M compared to native phospholipase A2, illustrating that the substrate specificity has changed enormously. The methionine substitutions were also prepared in a 69F mutant in which a part of the surface loop (residues 62–66) was deleted. Also in this deletion mutant these substitutions showed a similar effect as the substitutions in the native 69F mutant. Furthermore it was shown that deletion of the loop increases the activity on micellar lecithins and negatively charged micellar substrates, but reduces the activity on Lau2GroMe2P. Therefore it can be concluded that the loop is important for the recognition of substrates. We also show that the loop plays a role in the dimerization of these proteins. Dimerization may account for the high activities observed for some mutants acting on monomeric substrate. [ABSTRACT FROM AUTHOR]