Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology最新文献

{"title":"Cloning of human 3-hydroxyanthranilic acid dioxygenase in Escherichia coli: characterisation of the purified enzyme and its in vitro inhibition by Zn2+","authors":"Vito Calderone ,&nbsp;Michela Trabucco ,&nbsp;Valentina Menin ,&nbsp;Alessandro Negro ,&nbsp;Giuseppe Zanotti","doi":"10.1016/S0167-4838(02)00216-9","DOIUrl":"10.1016/S0167-4838(02)00216-9","url":null,"abstract":"<div><p>3-Hydroxyanthranilic acid oxygenase (3-HAO) catalyses the conversion of 3-hydroxyanthranilic acid to quinolinic acid. Because of the involvement of quinolinic acid in the initiation of neurodegenerative phenomena, we have cloned human 3-HAO in <em>Escherichia coli</em>, overexpressed and purified it with the aim of studying its enzymatic activity and for future structural studies. The recombinant human protein, obtained in <em>E. coli</em>, retains its enzymatic activity which can occur only in the presence of Fe(II); several other metals have been tested but in no case the formation of the product has been observed. On the contrary, two of the ions tested inhibit the catalytic reaction and one of them, Zn²⁺, could be of physiological relevance. A circular dichroism analysis has also been performed, showing that the secondary structure is mainly of the β type, with a minority of α.</p></div>","PeriodicalId":100166,"journal":{"name":"Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2002-04-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/S0167-4838(02)00216-9","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"123142546","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Calf spleen purine nucleoside phosphorylase: complex kinetic mechanism, hydrolysis of 7-methylguanosine, and oligomeric state in solution","authors":"Agnieszka Bzowska","doi":"10.1016/S0167-4838(02)00218-2","DOIUrl":"10.1016/S0167-4838(02)00218-2","url":null,"abstract":"<div><p>The active enzyme form was found to be a homotrimer, no active monomers were observed. Only in the presence of an extremely high orthophosphate concentration (0.5 M) or at a low enzyme concentration (0.2 μg/ml) with no ligands present a small fraction of the enzyme is probably in a dissociated and/or non-active form. The specific activity is invariant over a broad enzyme concentration range (0.017 μg/ml–0.29 mg/ml). At concentrations below 0.9 μg/ml and in the absence of ligands the enzyme tends to loose its catalytic activity, while in the presence of any substrate or at higher concentrations it was found to be active as a trimer. In the absence of phosphate the enzyme catalyses the hydrolysis of 7-methylguanosine (m⁷Guo) with a catalytic rate constant 1.3×10⁻³ s⁻¹ as compared with the rate of 38 s⁻¹ for the phosphorolysis of this nucleoside. The initial pre-steady-state phase of the phosphorolysis of m⁷Guo, 70 s⁻¹, is almost twice faster than the steady-state rate and indicates that the rate-limiting step is subsequent to the glycosidic bond cleavage. Complex kinetic behaviour with substrates of phosphorolytic direction (various nucleosides and orthophosphate) was observed; data for phosphate as the variable substrate with inosine and guanosine, but not with their 7-methyl counterparts, might be interpreted as two binding sites with different affinities, or as a negative cooperativity. However, the titration of the enzyme intrinsic fluorescence with 0.2 μM–30 mM phosphate is consistent with only one dissociation constant for phosphate, <em>K</em>_d=220±120 μM. Protective effects of ligands on the thermal inactivation of the enzyme indicate that all substrates of the phosphorolytic and the synthetic reactions are able to form binary complexes with the calf spleen purine nucleoside phosphorylase. The purine bases, guanine and hypoxanthine, bind strongly with dissociation constants of about 0.1 μM, while all other ligands studied, including 7-methylguanine and 7-methylhypoxanthine, bind at least 3 orders of magnitude less potently. Binding of guanine and hypoxanthine is about 10-fold weakened by the presence of phosphate. These observations are best interpretable by the complex kinetic mechanism of the phosphorolytic reaction involving (i) random substrate binding, (ii) unusually slow, hence strongly rate-limiting, dissociation of the products guanine and hypoxanthine, but not 7-methylguanine and 7-methylhypoxanthine, and (iii) dual function of the phosphate binding site with phosphate acting as a substrate and as a modifier helping in the release of a purine base after glycosidic bond cleavage.</p></div>","PeriodicalId":100166,"journal":{"name":"Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2002-04-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/S0167-4838(02)00218-2","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"74682631","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The surface exposed amino acid residues of monomeric proteins determine the partitioning in aqueous two-phase systems","authors":"Kristina Berggren ,&nbsp;Alejandro Wolf ,&nbsp;Juan A. Asenjo ,&nbsp;Barbara A. Andrews ,&nbsp;Folke Tjerneld","doi":"10.1016/S0167-4838(02)00222-4","DOIUrl":"10.1016/S0167-4838(02)00222-4","url":null,"abstract":"<div><p><span>It is of great interest and importance how different amino acid residues contribute to and affect the properties of a protein surface. Partitioning in aqueous two-phase systems has the potential to be used as a rapid and simple method for studying the surface properties of proteins. The influence on partitioning of the surface exposed amino acid residues of eight structurally determined monomeric proteins has been studied. The proteins were characterized in terms of surface exposed residues with a computer program, Graphical Representation and Analysis of Surface Properties (GRASP), and partitioned in two EO30PO70–dextran aqueous two-phase systems, only differing in polymer concentrations (system I: 6.8% EO30PO70, 7.1% dextran; system II: 9% EO30PO70, 9% dextran). We show for the first time that the partitioning behaviour of different monomeric proteins can be described by the differences in surface exposed amino acid residues. The contribution to the partition coefficient of the residues was found to be best characterized by peptide partitioning in the aqueous two-phase system. Compared to hydrophobicity scales available in the literature, each amino acid contribution is characterized by the slope given by the graph of log </span><em>K</em> against peptide chain length, for peptides of different length containing only one kind of residue. It was also shown that each amino acid contribution is relative to the total protein surface and the other residues on the surface. Surface hydrophobicity calculations realized for systems I and II gave respectively correlation coefficients of 0.961 and 0.949 for the linear relation between log <em>K</em> and calculated hydrophobicity values. To study the effect on the partition coefficient of different amino acids, they were grouped into classes according to common characteristics: the presence of an aromatic group, a long aliphatic chain or the presence of charge. Using these groups it was possible to confirm that aromatic residues have the strongest effect on the partition coefficient, giving preference to the upper EO30PO70 phase of the system; on the other hand the presence of charged amino acids on the protein surface enhances the partition of the protein to the lower dextran phase. It is also important to note that the sensitivity of the EO30PO70–dextran system for the surface exposed residues was increased by increasing the polymer concentrations. The partition coefficient of a monomeric protein can thus be predicted from its surface exposed amino acid residues and the system can also be used to characterize protein surfaces of monomeric proteins in general.</p></div>","PeriodicalId":100166,"journal":{"name":"Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2002-04-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/S0167-4838(02)00222-4","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"77619951","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Enhancement of plasminogen activation by surfactin C: augmentation of fibrinolysis in vitro and in vivo","authors":"Tadashi Kikuchi,&nbsp;Keiji Hasumi","doi":"10.1016/S0167-4838(02)00221-2","DOIUrl":"10.1016/S0167-4838(02)00221-2","url":null,"abstract":"<div><p>The reciprocal activation of plasminogen and prourokinase (pro-u-PA) is an important mechanism in the initiation and propagation of local fibrinolytic activity. We have found that a bacterial lipopeptide compound, surfactin C (3–20 μM), enhances the activation of pro-u-PA in the presence of plasminogen. This effect accompanied increased conversions of both pro-u-PA and plasminogen to their two-chain forms. Surfactin C also elevated the rate of plasminogen activation by two-chain urokinase (tcu-PA) while not affecting plasmin-catalyzed pro-u-PA activation and amidolytic activities of tcu-PA and plasmin. The intrinsic fluorescence of plasminogen was increased, and molecular elution time of plasminogen in size-exclusion chromatography was shortened in the presence of surfactin C. These results suggested that surfactin C induced a relaxation of plasminogen conformation, thus leading to enhancement of u-PA-catalyzed plasminogen activation, which in turn caused feedback pro-u-PA activation. Surfactin C was active in enhancing [¹²⁵I]fibrin degradation both by pro-u-PA/plasminogen and tcu-PA/plasminogen systems. In a rat pulmonary embolism model, surfactin C (1 mg/kg, i.v.) elevated ¹²⁵I plasma clot lysis when injected in combination with pro-u-PA. The present results provide first evidence that pharmacological relaxation of plasminogen conformation leads to enhanced fibrinolysis in vivo.</p></div>","PeriodicalId":100166,"journal":{"name":"Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2002-04-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/S0167-4838(02)00221-2","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"89631798","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Crystallization and preliminary X-ray crystallographic analysis of Ace: a Collagen-binding MSCRAMM from Enterococcus faecalis","authors":"Karthe Ponnuraj ,&nbsp;Yi Xu ,&nbsp;Dwight Moore ,&nbsp;Champion C.S. Deivanayagam ,&nbsp;Lluis Boque ,&nbsp;Magnus Hook ,&nbsp;Sthanam V.L. Narayana","doi":"10.1016/S0167-4838(01)00328-4","DOIUrl":"10.1016/S0167-4838(01)00328-4","url":null,"abstract":"<div><p>Ace is a collagen-binding bacterial cell surface adhesin from <em>Enterococcus faecalis</em>. The collagen-binding domain of Ace (termed Ace40) and its truncated form Ace19 have been crystallized by the vapor-diffusion hanging-drop method. Ace19 was crystallized in two different crystal forms. A complete 1.65 Å data set has been collected on the orthorhombic crystal form with unit cell parameters <em>a</em>=38.43 <em>b</em>=48.91 and <em>c</em>=83.73 Å. Ace40 was crystallized in the trigonal space group P3₁21 or P3₂21 with unit cell parameters <em>a</em>=<em>b</em>=80.24, <em>c</em>=105.91 Å; <em>α</em>=<em>β</em>=90 and <em>γ</em>=120°. A full set of X-ray diffraction data was collected to 2.5 Å. Three heavy atom derivative data sets have been successfully obtained for Ace19 crystals and structural analysis is in progress.</p></div>","PeriodicalId":100166,"journal":{"name":"Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2002-04-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/S0167-4838(01)00328-4","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"83567335","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Crystal structure of the double azurin mutant Cys3Ser/Ser100Pro from Pseudomonas aeruginosa at 1.8 Å resolution: its folding–unfolding energy and unfolding kinetics","authors":"Mats Ökvist ,&nbsp;Nicklas Bonander ,&nbsp;Anders Sandberg ,&nbsp;B.Göran Karlsson ,&nbsp;Ute Krengel ,&nbsp;Yafeng Xue ,&nbsp;Lennart Sjölin","doi":"10.1016/S0167-4838(02)00215-7","DOIUrl":"10.1016/S0167-4838(02)00215-7","url":null,"abstract":"<div><p>Azurin is a cupredoxin, which functions as an electron carrier. Its fold is dominated by a β-sheet structure. In the present study, azurin serves as a model system to investigate the importance of a conserved disulphide bond for protein stability and folding/unfolding. For this purpose, we have examined two azurin mutants, the single mutant Cys3Ser, which disrupts azurin’s conserved disulphide bond, and the double mutant Cys3Ser/Ser100Pro, which contains an additional mutation at a site distant from the conserved disulphide. The crystal structure of the azurin double mutant has been determined to 1.8 Å resolution<span>²</span>, with a crystallographic <em>R</em>-factor of 17.5% (<em>R</em>_free=20.8%). A comparison with the wild-type structure reveals that structural differences are limited to the sites of the mutations. Also, the rates of folding and unfolding as determined by CD and fluorescence spectroscopy are almost unchanged. The main difference to wild-type azurin is a destabilisation by ∼20 kJ mol⁻¹, constituting half the total folding energy of the wild-type protein. Thus, the disulphide bond constitutes a vital component in giving azurin its stable fold.</p></div>","PeriodicalId":100166,"journal":{"name":"Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2002-04-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/S0167-4838(02)00215-7","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"76589286","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"New aspects on the mechanism of GroEL-assisted protein folding","authors":"Petra Guhr ,&nbsp;Sonja Neuhofen ,&nbsp;Carol Coan ,&nbsp;John G. Wise ,&nbsp;Pia D. Vogel","doi":"10.1016/S0167-4838(02)00219-4","DOIUrl":"10.1016/S0167-4838(02)00219-4","url":null,"abstract":"<div><p>The mechanism of assisted protein folding by the chaperonin GroEL alone or in complex with the co-chaperonin GroES and in the presence or absence of nucleotides has been subject to extensive investigations during the last years. In this paper we present data where we have inactivated GroEL by stepwise blocking the nucleotide binding sites using the non-hydrolyzable ATP analogue, (Cr(H₂O)₄)³⁺ATP. We correlated the amount of accessible nucleotide binding sites with the residual ATP hydrolysis activity of GroEL as well as the residual refolding activity for two different model substrates. Under the conditions used, folding of the substrate proteins and ATP hydrolysis were directly proportional to the residual, accessible nucleotide binding sites. In the presence of GroES, 50% of the nucleotide binding sites were protected from inactivation by CrATP and the resulting protein retains 50% of both ATPase and refolding activity. The results strongly suggest that under the conditions used in our experiments, the nucleotide binding sites are additive in character and that by blocking of a certain number of binding sites a proportional amount of ATP hydrolysis and refolding activities are inactivated. The experiments including GroES suggest that full catalytic activity of GroEL requires both rings of the chaperonin. Blocking of the nucleotide binding sites of one ring still allows function of the second ring.</p></div>","PeriodicalId":100166,"journal":{"name":"Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2002-04-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/S0167-4838(02)00219-4","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"80839629","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Molecular and functional properties of an archaeal phenylalanyl-tRNA synthetase from the hyperthermophile Sulfolobus solfataricus","authors":"Barbara Lombardo,&nbsp;Gennaro Raimo,&nbsp;Vincenzo Bocchini","doi":"10.1016/S0167-4838(02)00223-6","DOIUrl":"10.1016/S0167-4838(02)00223-6","url":null,"abstract":"<div><p>An archaeal phenylalanyl-tRNA synthetase (FRS) has been purified from the hyperthermophile <em>Sulfolobus solfataricus</em> (<em>Ss</em>). This enzyme is a heterotetramer made of two different subunits whose molecular mass is 56 kDa and 64 kDa, respectively. As thought, <em>Ss</em>FRS is essential for the in vitro poly(Phe) synthesis. Interestingly, the enzyme is able to aminoacylate only endogenous tRNA but it does not seem to be a strictly ATP-dependent synthetase. <em>Ss</em>FRS interacts with the elongation factor 1α isolated from the same source; this caused a significant enhancement of the <em>Ss</em>tRNA aminoacylation efficiency, thus indicating that, as well as in eukarya, in this archaeon a tRNA channelling mechanism should occur. The overall results presented in this paper show that the archaeal <em>Ss</em>FRS behaves as the analogous enzymes isolated from eukaryal sources rather than those from eubacterial organisms.</p></div>","PeriodicalId":100166,"journal":{"name":"Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2002-04-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/S0167-4838(02)00223-6","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"89209306","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Kinetic analysis of mouse retinal dehydrogenase type-2 (RALDH2) for retinal substrates","authors":"Isabelle Gagnon ,&nbsp;Gregg Duester ,&nbsp;Pangala V. Bhat","doi":"10.1016/S0167-4838(02)00213-3","DOIUrl":"10.1016/S0167-4838(02)00213-3","url":null,"abstract":"<div><p>Retinal dehydrogenase (RALDH) isozymes catalyze the terminal oxidation of retinol into retinoic acid (RA) that is essential for embryogenesis and tissue differentiation. To understand the role of mouse type 2 RALDH in synthesizing the ligands (all-<em>trans</em> and 9-<em>cis</em> RA) needed to bind and activate nuclear RA receptors, we determined the detailed kinetic properties of RALDH2 for various retinal substrates. Purified recombinant RALDH2 showed a pH optimum of 9.0 for all-<em>trans</em> retinal oxidation. The activity of the enzyme was lower at 37°C compared to 25°C. The efficiency of conversion of all-<em>trans</em> retinal to RA was 2- and 5-fold higher than 13-<em>cis</em> and 9-<em>cis</em> retinal, respectively. The <em>K</em>_m for all-<em>trans</em> and 13-<em>cis</em> retinal were similar (0.66 and 0.62 μM, respectively). However, the <em>K</em>_m of RALDH2 for 9-<em>cis</em> retinal substrate (2.25 μM) was 3-fold higher compared to all-<em>trans</em> and 13-<em>cis</em> retinal substrates. Among several reagents tested for their ability to either inhibit or activate RALDH2, citral and <em>para</em>-hydroxymercuribenzoic acid (p-HMB) inhibited and MgCl₂ activated the reaction. Comparison of the kinetic properties of RALDH2 for retinal substrates and its activity towards various reagents with those of previously reported rat kidney RALDH1 and human liver aldehyde dehydrogenase-1 showed distinct differences. Since RALDH2 has low <em>K</em>_m and high catalytic efficiency for all-<em>trans</em> retinal, it may likely be involved in the production of all-<em>trans</em> RA in vivo.</p></div>","PeriodicalId":100166,"journal":{"name":"Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2002-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/S0167-4838(02)00213-3","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"80354703","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Upward shift of the pH optimum of Acremonium ascorbate oxidase","authors":"Masayasu Sugino ,&nbsp;Sachiko Kajita ,&nbsp;Koichi Banno ,&nbsp;Tsuyoshi Shirai ,&nbsp;Takashi Yamane ,&nbsp;Masashi Kato ,&nbsp;Tetsuo Kobayashi ,&nbsp;Norihiro Tsukagoshi","doi":"10.1016/S0167-4838(01)00310-7","DOIUrl":"10.1016/S0167-4838(01)00310-7","url":null,"abstract":"<div><p>A gene encoding a thermostable <em>Acremonium</em> ascorbate oxidase (ASOM) was randomly mutated to generate mutant enzymes with altered pH optima. One of the mutants, which exhibited a significantly higher activity in the pH range 4.5–7 compared to ASOM, had a Gln183Arg substitution in the region corresponding to SBR1, one of the substrate binding regions of the zucchini enzyme. The other mutant with almost the same pH profile as Gln183Arg had a Thr527Ala substitution near the type 3 copper center and became more sensitive to azide than ASOM. Site-directed mutagenesis in the substrate binding regions with reference to the amino acid sequences of plant enzymes led to isolation of mutants shifted upward in the pH optimum; Val193Pro and Val193Pro/Pro190Ile increased the pH optimum by 1 and 0.5 units, respectively, while retaining the near-wild-type thermostability and azide sensitivity. The homology model of ASOM constructed from the zucchini enzyme coordinates suggested that replacement of Val193 by Pro could disturb the ion pair networks among Arg309, Glu192, Arg194 and Glu311. This perturbation could affect either the molecular recognition between the substrate and ASOM or the electron transfer from the substrate to the type 1 copper center, leading to the alkaline shift of the catalytic activity of the mutant enzyme. The other mutations, Val193Pro/Pro190Ile, could also induce similar structural perturbations involving the ion pair networks.</p></div>","PeriodicalId":100166,"journal":{"name":"Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2002-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/S0167-4838(01)00310-7","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"78884173","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}