Isolation and characterization of active N-terminal truncated apo- and holoenzyme of mammalian liver tyrosine aminotransferase.

Limited proteolysis was used to probe the structure of the apo- and holoenzyme of rat liver tyrosine aminotransferase. Both were subjected to trypsinolysis and the major fragments were isolated and characterized. Trypsin cleaves the apoenzyme after residues Arg57, Lys64, and Lys71 and the holoenzyme after Arg37 and Lys38. The difference in the accessibility of the enzyme deprived or associated with pyridoxal 5'-phosphate reflects two distinct conformations. The activity, the affinity for the ligands and the thermostability of the purified truncated enzyme forms are similar to those of the native apo- and holoenzyme. A model for the domain structure of mammalian tyrosine aminotransferase and a mechanism for its rapid turnover are proposed.     

Topography of prostaglandin H synthase. Antiinflammatory agents and the protease-sensitive arginine 253 region

Prostaglandin H synthase catalyzes two reactions: the bis-dioxygenation of arachidonic acid to form prostaglandin G2 (cyclooxygenase activity), and the reduction of hydroperoxides to the corresponding alcohols (peroxidase activity). The cyclooxygenase activity can be selectively inhibited by many nonsteroidal antiinflammatory agents including indomethacin. In the native synthase, there is a single prominent protease-sensitive region, located near Arg253; binding of the heme prosthetic group makes the synthase resistant to proteases. To investigate the spatial relationship between the area of the synthase which interacts with indomethacin and the protease-sensitive region, the effects of indomethacin and similar agents on the protease sensitivity of the two enzymatic activities and of the synthase polypeptide were examined. Incubation of the synthase apoenzyme with trypsin (3.6% w/w) resulted in the time-dependent coordinate loss (75% at 1 h) of both enzymatic activities and the cleavage (85% at 1 h) of the 70-kDa subunit into 38- and 33-kDa fragments, indicating that proteolytic cleavage of the polypeptide at Arg253, destroyed both activities of the synthase simultaneously. Indomethacin, (S)-flurbiprofen, or meclofenamate (each at 20 microM) rendered both activities and the synthase polypeptide (at 5 microM subunit) resistant to attack by trypsin or proteinase K; these agents also inhibited the cyclooxygenase activity of the intact synthase. Two reversible cyclooxygenase inhibitors, ibuprofen and flufenamate, also made both of the activities and the synthase polypeptide more resistant to trypsin. Titration of the apoenzyme with indomethacin (0-3 mol/mol of synthase dimer) resulted in proportional increases in the inhibition of the cyclooxygenase and in the resistance to attack by trypsin. (R)-Flurbiprofen did not increase the resistance to protease or appreciably inhibit the cyclooxygenase. These results suggest that the same stereospecific interaction of these agents with the synthase that produced inhibition of the cyclooxygenase led to a decreased accessibility of the Arg253 region to proteases. Aspirin treatment made the synthase less resistant to trypsin; aspirin-treated synthase became more resistant to trypsin when it was incubated with indomethacin before addition of the protease. The presence of 50 microM arachidonate during digestion of apoenzyme or aspirin-treated apoenzyme with trypsin did not decrease the cleavage of the synthase subunit.(ABSTRACT TRUNCATED AT 400 WORDS)     

Stability and Activation of Glutamate Apodecarboxylase from Pig Brain

The stability and activation of glutamate apodecarboxylase was studied with three forms of the enzyme from pig brain (referred to as the alpha, beta, and gamma forms). Apoenzyme was prepared by incubating the holoenzyme with aspartate followed by chromatography on Sephadex G-25. Apoenzyme was much less stable than holoenzyme to inactivation by heat (for beta-glutamate decarboxylase (beta-GAD) at 30 degrees C, t1/2 values of apo- and holoenzyme were 17 and greater than 100 min). ATP protected holoenzyme and apoenzyme against heat inactivation. The kinetics of reactivation of apoenzyme by pyridoxal-P was consistent with a two-step mechanism comprised of a rapid, reversible association of the cofactor with apoenzyme followed by a slow conversion of the complex to active holoenzyme. The reactivation rate constant (kr) and apparent dissociation constant (KD) for the binding of pyridoxal-P to apoenzyme differed substantially among the forms (for alpha-, beta-, and gamma-GAD, kr = 0.032, 0.17, and 0.27 min-1, and KD = 0.014, 0.018, and 0.04 microM). ATP was a strong competitive inhibitor of activation (Ki = 0.45, 0.18, and 0.39 microM for alpha-, beta-, and gamma-GAD). In contrast, Pi stimulated activation at 1-5 mM but inhibited at much higher concentrations. The results suggest that ATP is important in stabilizing the apoenzyme in brain and that ATP, Pi, and other compounds regulate its activation.     

Effect of Cytokinin on the Enhancement of Nitrate Reductase NR Activity in Tobacco Callus Tissues and Wheat Seedlings

The effect of cytokinin on the formation of NR activity were studied with tobacco callus tissues and wheat seedlings. Cytokinin could not induce the NR activity alone but could enhance the NR inducibility (Table 1). The enhancement of NR formation was detected in the tissues pretreated with cytokinin for over 12 hours. It showed that there was a precondition in the tissues for the induction of NR (Fig. 3). The precondition could not be improved by cytokinin when cycloheximide (inhibitor of protein synthesis) was added into the medium during cytokinin pretreatment (Table 2). Thus, it was thought that cytokinin might enhance synthesis of a protein which participated in the NR activity induction. In immunological test (Fig. 5) the existence of a nonactive apoenzyme of NR in higher plant tissues was demonstrated. It is, therefore, suggested that there are two major steps in the NR activity formation: (l) the synthesis of a nonactive NR apoenzyme, (2) the activation of this nonactive apoenzyme. The former step might be stimulated by cytokinin and the latter was mediated by nitrate.     

Biochemical and biological properties of DNA photolyases derived from utraviolet-sensitive rice cultivars

Class I and class II CPD photolyases are enzymes which repair pyrimidine dimers using visible light. A detailed characterization of class I CPD photolyases has been carried out, but little is known about the class II enzymes. Photolyases from rice are suitable for functional analyses because systematic breeding for long periods in Asian countries has led to the selection of naturally occurring mutations in the CPD photolyase gene. We report the biochemical characterization of rice mutant CPD photolyases purified as GST-form from Escherichia coli. We identified three amino acid changes, Gln126Arg, Gly255Ser, and Gln296His, among which Gln but not His at 296 is important for complementing phr-defective E. coli, binding UV-damage in E. coli, and binding thymine dimers in vitro. The photolyase with Gln at 296 has an apoenzyme:FAD ratio of 1 : 0.5 and that with His at 296 has an apoenzyme:FAD ratio of 1 : 0.12-0.25, showing a role for Gln at 296 in the binding of FAD not in the binding of thymine dimer. Concerning Gln or Arg at 126, the biochemical activity of the photolyases purified from E. coli and complementing activity for phr-defective E. coli are similarly proficient. However, the sensitivity to UV of cultivars differs depending on whether Gln or Arg is at 126. The role of Gln and Arg at 126 for photoreactivation in rice is discussed.     

Kinetics of the inactivation of Escherichia coli glutamate apodecarboxylase by phenylglyoxal.

The possible interaction of the phosphate moiety of pyridoxal phosphate with a guanidinium group in glutamate apodecarboxylase was investigated. The holoenzyme is not inactivated significantly by incubation with butanedione, glyoxal, methylglyoxal, or phenylglyoxal. However, the apoenzyme is inactivated by these arginine reagents in time-dependent processes. Phenylgloxal inactivates the apoenzyme most rapidly. The inactivation follows pseudo-first-order kinetics at high phenylglyoxal to apoenzyme ratios. The rate of inactivation is proportional to phenylglyoxal concentration, increases with increasing pH, and is also dependent on the type of buffer present. The rate of inactivation of the apoenzyme by phenylglyoxal is fastest in bicarbonate — carbonate buffer and increases with increasing bicarbonate — carbonate concentration. Phosphate, which inhibits the binding of pyridoxal phosphate to the apoenzyme, protects the apodecarboxylase against inactivation by phenylglyoxal. When the apodecarboxylase is inactivated with [¹⁴C]phenylglyoxal, approximately 1.6 mol of [¹⁴C]phenylglyoxal is incorporated per mol subunit. The phenylglyoxal is thought to modify an arginyl residue at or near the pyridoxal phosphate binding site of glutamate apodecarboxylase.     

The mutation of two amino acid residues in the N-terminus of tyrosine hydroxylase (TH) dramatically enhances the catalytic activity in neuroendocrine AtT-20 cells

The sequence Arg37-Arg38 of tyrosine hydroxylase (TH) is known to play a significant role in the feedback inhibition by the end product DA. To clarify how deeply the sequence Arg37-Arg38 and the phosphorylated Ser40 of human TH type 1 (hTH1) are involved in the regulation of this feedback inhibition in mammalian cells, we generated the following mutants: (i) RR-GG, Arg37-Arg38 replaced by Gly37-Gly38; (ii) RR-EE, Arg37-Arg38 replaced by Glu37-Glu38; (iii) S40D, Ser40 replaced by Asp40; and (iv) S40A, Ser40 replaced by Ala40. In a cell-free system, the level of the DA inhibition of the RR-EE mutant enzyme was to the same or smaller degree than that of the phosphorylation-mimicking S40D. Next, AtT-20 neuroendocrine cells were transfected with wild-type and mutated TH genes because these cells were earlier shown to be capable of fully converting L-3,4-dihydroxyphenylalanine into DA, whereby the catalytic activity of TH would be expected to be inhibited by the end product DA accumulating in the cells. The level of DA accumulation in AtT-20 cells expressing the TH gene was in the order: RR-EE > S40D > S40A = RR-GG > wild-type, which was in accordance with the observations for the cell-free system. These results suggest that the sequence Arg37-Arg38 of hTH1 is a more potent determinant of the efficient production of DA in mammalian cells than is the phosphorylated Ser40-hTH1.     

Structures of apo- and holo-tyrosine phenol-lyase reveal a catalytically critical closed conformation and suggest a mechanism for activation by K+ ions

Tyrosine phenol-lyase, a tetrameric pyridoxal 5'-phosphate dependent enzyme, catalyzes the reversible hydrolytic cleavage of L-tyrosine to phenol and ammonium pyruvate. Here we describe the crystal structure of the Citrobacter freundii holoenzyme at 1.9 A resolution. The structure reveals a network of protein interactions with the cofactor, pyridoxal 5'-phosphate, and details of coordination of the catalytically important K+ ion. We also present the structure of the apoenzyme at 1.85 A resolution. Both structures were determined using crystals grown at pH 8.0, which is close to the pH of the maximal enzymatic activity (8.2). Comparison of the apoenzyme structure with the one previously determined at pH 6.0 reveals significant differences. The data suggest that the decrease of the enzymatic activity at pH 6.0 may be caused by conformational changes in the active site residues Tyr71, Tyr291, and Arg381 and in the monovalent cation binding residue Glu69. Moreover, at pH 8.0 we observe two different active site conformations: open, which was characterized before, and closed, which is observed for the first time in beta-eliminating lyases. In the closed conformation a significant part of the small domain undergoes an extraordinary motion of up to 12 A toward the large domain, closing the active site cleft and bringing the catalytically important Arg381 and Phe448 into the active site. The closed conformation allows rationalization of the results of previous mutational studies and suggests that the observed active site closure is critical for the course of the enzymatic reaction and for the enzyme's specificity toward its physiological substrate. Finally, the closed conformation allows us to model keto(imino)quinonoid, the key transition intermediate.     

Mutation of residues critical for benzohydroxamic acid binding to horseradish peroxidase isoenzyme C.

Aromatic substrate binding to peroxidases is mediated through hydrophobic and hydrogen bonding interactions between residues on the distal side of the heme and the substrate molecule. The effects of perturbing these interactions are investigated by an electronic absorption and resonance Raman study of benzohydroxamic acid (BHA) binding to a series of mutants of horseradish peroxidase isoenzyme C (HRPC). In particular, the Phe179 --> Ala, His42 --> Glu variants and the double mutant His42 --> Glu:Arg38 --> Leu are studied in their ferric state at pH 7 with and without BHA. A comparison of the data with those previously reported for wild-type HRPC and other distal site mutants reaffirms that in the resting state mutation of His42 leads to an increase of 6-coordinate aquo heme forms at the expense of the 5-coordinate heme state, which is the dominant species in wild-type HRPC. The His42Glu:Arg38Leu double mutant displays an enhanced proportion of the pentacoordinate heme state, similar to the single Arg38Leu mutant. The heme spin states are insensitive to mutation of the Phe179 residue. The BHA complexes of all mutants are found to have a greater amount of unbound form compared to the wild-type HRPC complex. It is apparent from the spectral changes induced on complexation with BHA that, although Phe179 provides an important hydrophobic interaction with BHA, the hydrogen bonds formed between His42 and, in particular, Arg38 and BHA assume a more critical role in the binding of BHA to the resting state.     

Synthesis of pyruvate carboxylase from its apoenzyme and (+)-biotin in Bacillus stearothermophilus. Mechanism and control of the reaction

1. Acetyl-CoA acts as a positive allosteric effector in the formation of active pyruvate carboxylase from its apoenzyme, ATP and (+)-biotin which is catalysed by holoenzyme synthetase; this effect is counteracted by l-aspartate. 2. The Hill coefficients (apparent n values) were approximately 2 for acetyl-CoA and 4 for l-aspartate; the n value for each effector remained constant when the concentration of the other effector was varied. 3. Active pyruvate carboxylase was formed also when the apoenzyme was incubated with holoenzyme synthetase and synthetic biotinyl-5'-AMP; acetyl-CoA and l-aspartate affected this process as they did the overall reaction from (+)-biotin and ATP. 4. When hydroxylamine replaced the apoenzyme, holoenzyme synthetase catalysed the formation of biotinylhydroxamate from (+)-biotin and ATP. This reaction was not affected by the allosteric effectors. 5. The apoenzyme was protected against thermal denaturation by acetyl-CoA and, to a lesser degree, by l-aspartate. The holoenzyme synthetase was not markedly protected by these effectors. 6. It is concluded that the allosteric effectors act on the apoenzyme and not the synthetase.     

The conformational stability of the redox states of lipoamide dehydrogenase from Azotobacter vinelandii.

The conformational stability of holo-lipoamide and apo-lipoamide dehydrogenase from Azotobacter vinelandii was studied by thermoinactivation, unfolding and limited proteolysis. The oxidized holoenzyme is thermostable, showing a melting temperature, tm = 80 degrees C. The thermal stability of the holoenzyme drastically decreases upon reduction. Unlike the oxidized and lipoamide two-electron reduced enzyme species, the NADH four-electron reduced enzyme is highly sensitive to unfolding by urea. Loss of energy transfer from Trp199 to flavin reflects the unfolding of the oxidized holoenzyme by guanidine hydrochloride. Unfolding of the monomeric apoenzyme is a rapid fully reversible process, following a simple two-state mechanism. The oxidized and two-electron reduced holoenzyme are resistant to limited proteolysis by trypsin and endoproteinase Glu-C. Upon cleavage of the apoenzyme or four-electron reduced holoenzyme by both proteases, large peptide fragments (molecular mass greater than 40 kDa) are transiently produced. Sequence studies show that limited trypsinolysis of the NADH-reduced enzyme starts mainly at the C-terminus of Arg391. In the apoenzyme, limited proteolysis by endoproteinase Glu-C starts from the C-terminus at the carboxyl ends of Glu459 and/or Glu435. From crystallographic data it is deduced that the susceptible amino acid peptide bonds are situated near the subunit interface. Thus, these bonds are inaccessible to the proteases in the dimeric enzyme and become accessible after monomerization. It is concluded that reduction of lipoamide dehydrogenase to the four-electron reduced state(s) is accompanied by conformational changes promoting subunit dissociation.     

Structure of a triclinic ternary complex of horse liver alcohol dehydrogenase at 2.9 Å resolution

The structure of a triclinic complex between liver alcohol dehydrogenase, reduced coenzyme NADH, and the inhibitor dimethylsulfoxide has been determined to 2.9 Å resolution using isomorphous replacement methods. The heavy-atom positions were derived by molecular replacement methods using phase angles derived from a model of the orthorhombic apoenzyme structure previously determined to 2.4 Å resolution. A model of the present holoenzyme molecule was built on a Vector General 3400 display system using the RING system of programs. This model gave a crystallographic R-value of 37.9%.There are extensive conformational differences between the protein molecules in the two forms. The conformational change involves a rotation of 7.5 ° of the catalytic domains relative to the coenzyme binding domains. A hinge region for this rotation is defined within a hydrophobic core between two helices. The internal structures of the domains are preserved with the exception of a movement of a small loop in the coenzyme binding domain. A cleft between the domains is closed by this coenzyme-induced conformational change, making the active site less accessible from solution and thus more hydrophobic.The two crystallographically independent subunits are very similar and bind both coenzyme and inhibitor in an identical way within the present limits of error. The coenzyme molecule is bound in an extended conformation with the two ends in hydrophobic crevices on opposite sides of the central pleated sheet of the coenzyme binding domain. There are hydrogen bonds to oxygen atoms of the ribose moities from Asp223, Lys228 and His51. The pyrophosphate group is in contact with the side-chains of Arg47 and Arg369.No new residues are brought into the active site compared to the apoenzyme structure. The active site zinc atom is close to the hinge region, where the smallest structural changes occur. Small differences in the co-ordination geometry of the ligands Cys46, His67 and Cysl74 are not excluded and may account for the ordered mechanism. The oxygen atom of the inhibitor dimethylsulfoxide is bound directly to zinc confirming the structural basis for the suggested mechanism of action based on studies of the apoenzyme structure.     

Bromopyruvate inactivation of glutamate apodecarboxylase. Kinetics and specificity.

A number of halo carboxylic and dicarboxylic acids were substrate-competitive inhibitors of glutamate decarboxylase, with bromosuccinate, 3-bromopropionate, and iodoacetate having the highest affinity for the enzyme. Some of the halo acids also inactivated the apoenzyme. Bromopyruvate at relatively low concentrations inactivated the apoenzyme irreversibly. The rate of the inactivation of the apodecarboxylase was proportional to bromopyruvate at low concentration and approached a constant rate of inactivation at high bromopyruvate concentration. These data are consistent with a two-step inactivation process in which an enzyme-bromopyruvate complex is formed followed by inactivation. The concentration of bromopyruvate giving the half-maximum rate of inactivation was 6.9 mM, and the maximum rate of inactivation was 1.75 min-1 at pH 4.6 and 23 degrees. Much faster rates of inactivation were obtained at pH 5.96 and 6.44. Phosphate, an inhibitor of pyrisoxal-P binding to the apoenzyme, competitively inhibited the inactivation of the apoenzyme by bromopyruvate. In addition, bromopyruvate inhibited the rate of pyridoxal-P binding to the apoenzyme. Kinetics of the incorporation of bromo[2-14C]pyruvate indicated that complete inactivation was obtained when 1.2 mol of radioactive residue were covalently bound per subunit of apoenzyme. Amino acid analyses demonstrated that a cysteinyl residue was alkylated by the bromopyruvate. The bromopyruvate was evidently interacting nincovalently with a cationic group at or near the pyridoxal-P-binding site, and then was alkylating a nearby cysteinyl residue.     

Studies on new intracellular proteases in various organs of rat. 2. Mode of limited proteolysis.

1. The mechanism of proteolysis of ornithine transaminase apoenzyme II by group-specific protease and the relation between the confirmations of ornithine transaminase and its susceptibility to group-specific protease were studied to elucidate the mode of action of the protease. 2. Differences in the conformations of ornithine transaminase apoenzyme II, molecular weight 67000, and ornithine transaminase holoenzyme, molecular weight 140000, were shown by studies on difference spectra produced by various concentrations of ethylene glycol. Increase of the titratable sulfhydryl groups on resolution of the coenzyme from ornithine transaminase also supports this finding. These results are consistent with the facts that the apoenzyme was sensitive to group-specific protease, while the holoenzyme was not. 3. Kinetics studies showed that ornithine transaminase apoenzyme II was degraded by limited proteolysis. Reaction of the native enzyme with group-specific protease resulted in a nick in the enzyme molecule with formation of one homogeneous large product and small peptides. The large product was not degraded further. The large product was indistinguishable from native ornithine transaminase apoenzyme II in various properties including its elution volume on gel filtration, its mobility on disc electrophoresis, its antigenicity, its estimated number of exposed tryptophan residues, and its titratable number of sulfhydryl groups. But unlike the apoenzyme the product did not show tetramerization with coenzyme or catalytic activity, although it retained the ability to bind with coenzyme and had the same number of bound pyridoxal phosphate as the native ornithine transaminase molecule. Thus, native ornithine transaminase apoenzyme II was degraded by limited proteolysis. Unfolded enzyme, denatured by 8 M urea, was degraded extensively. 4. The initial step of intracellular proteins degradation is discussed on the basis of these results.     

Contribution of active site residues to the activity and thermal stability of ribonuclease Sa

We have used site-specific mutagenesis to study the contribution of Glu 74 and the active site residues Gln 38, Glu 41, Glu 54, Arg 65, and His 85 to the catalytic activity and thermal stability of ribonuclease Sa. The activity of Gln38Ala is lowered by one order of magnitude, which confirms the involvement of this residue in substrate binding. In contrast, Glu41Lys had no effect on the ribonuclease Sa activity. This is surprising, because the hydrogen bond between the guanosine N1 atom and the side chain of Glu 41 is thought to be important for the guanine specificity in related ribonucleases. The activities of Glu54Gln and Arg65Ala are both lowered about 1000-fold, and His85Gln is totally inactive, confirming the importance of these residues to the catalytic function of ribonuclease Sa. In Glu74Lys, k(cat) is reduced sixfold despite the fact that Glu 74 is over 15 A from the active site. The pH dependence of k(cat)/K(M) is very similar for Glu74Lys and wild-type RNase Sa, suggesting that this is not due to a change in the pK values of the groups involved in catalysis. Compared to wild-type RNase Sa, the stabilities of Gln38Ala and Glu74Lys are increased, the stabilities of Glu41Lys, Glu54Gln, and Arg65Ala are decreased and the stability of His85Gln is unchanged. Thus, the active site residues in the ribonuclease Sa make different contributions to the stability.     

Inhibition mechanism of a peanut trypsin-chymotrypsin inhibitor, B-III: determination of the reactive sites for trypsin and chymotrypsin

Peanut inhibitor B-III was found to form two types of complexes with trypsin, T2I and TI, by gel filtration HPLC. Two cleaved peptide bonds, Arg(10)-Arg(11) and Arg(38)-Ser(39), in the trypsin modified inhibitor (TM-B-III*R*S) (J. Biochem. 93, 479-485 (1983] were resynthesized by the complex formation with 2 mol of trypsin. These results suggest that the two peptide bonds may be the reactive sites for trypsin. TM-B-III*R*S inhibited bovine trypsin as well as native B-III but had little chymotrypsin inhibitory activity. The two peptide bonds, Arg(10)-Arg(11) and Arg(38)-Ser(39), in B-III were cleaved partly by prolonged incubation with a catalytic amount of chymotrypsin. But gel filtration HPLC of the chymotrypsin-inhibitor complex showed the formation of only CI complex. Incubation of TM-B-III*R*S with an equimolar amount of chymotrypsin resulted in the resynthesis of only the Arg(10)-Arg(11) bond. These findings suggest that Arg(10)-Arg(11) may be a true reactive site for chymotrypsin. An inhibition mechanism of B-III against trypsin and chymotrypsin was proposed from the results obtained by the present studies.     

Instability of the apo form of aromatic L-amino acid decarboxylase in vivo and in vitro: implications for the involvement of the flexible loop that covers the active site

Pyridoxine deficiency caused a decrease in the amount of aromatic L-amino acid decarboxylase (AADC) in PC12 cells to less than 5% of the control. The degree of the enzyme saturation with the coenzyme pyridoxal 5'-phosphate (PLP) was around 90% for both the control and the pyridoxine-deficient cells, contrary to earlier reports by others. Mathematical analysis of the result indicated that the AADC apoenzyme is degraded at least 20-fold faster than the holoenzyme in the cells. To determine the mechanism of the preferential degradation of the apoenzyme, in vitro model studies were carried out. AADC has a flexible loop that covers the active site. This loop was easily leaved by proteases at similar rates for both the holoenzyme and the apoenzyme. However, in the presence of the substrate analog, dopa methyl ester, the holoenzyme was not cleaved by proteases, while the apoenzyme was cleaved similarly. These results indicated that the ligand that forms a Schiff base (aldimine) with PLP is fixed to the active site and stabilizes the flexible loop. The structure of the rat AADC-dopa complex modeled on the crystal structure of pig AADC showed that the flexible loop can fit in the concave surface at the entrance of the active site, its aliphatic and aromatic residues forming hydrophobic interactions with the substrate catechol ring. It was postulated that the flexible loop of the holoenzyme is stabilized in vivo by taking a closed structure that holds the PLP-substrate aldimine, while the apoenzyme cannot bind the substrate and its flexible loop is easily cleaved, leading to the preferential degradation of the apoenzyme.     

Heterologous high level expression, purification, and enzymological properties of recombinant rat cobalamin-dependent methionine synthase

Rat methionine synthase was expressed chiefly as apoenzyme in recombinant baculovirus-infected insect cells (Yamada, K., Tobimatsu, T., and Toraya, T. (1998) Biosci. Biotech. Biochem. 62, 2155-2160). The apoenzyme produced was very unstable, and therefore, after complexation with methylcobalamin, the functional holoenzyme was purified to homogeneity. The specific activity and apparent K(m) values for substrates were in good agreement with those obtained with purified rat liver enzyme. The electronic spectrum of the purified recombinant enzyme resembled that of cob(II)alamin and changed to a methylcobalamin-like one upon incubation of the enzyme with titanium(III) and S-adenosylmethionine. The rate of oxidative inactivation of the enzyme in the absence of S-adenosylmethionine was slower with a stronger reducing agent like titanium(III). The nucleotide moiety, especially the phosphodiester group, was shown to play an important role in the binding of the coenzyme to apoprotein and thus for catalysis. Upon incubation with the apoenzyme in the absence of a reducing agent, cyano- and aquacobalamin were not effective or were effective only slightly in reconstituting holoenzyme. Ethyl- and propylcobalamin formed inactive complexes with apoenzyme, which were converted to holoenzyme by photolytic activation. Adenosylcobalamin was not able to form a complex with apoenzyme, which was convertible to holoenzyme by photoirradiation.     

棕色固氮菌含铁超氧化物歧化酶重组研究

将棕色固氮菌230含铁超氧化物歧化酶对8mol/L脲,10mmol/L EDTA透析制备无活性缺辅基蛋白;将其在8mol/L脲中对10mmol/L硫酸亚铁铵透析得到重组超氧化物歧化酶。重组酶含有与天然酶相近的铁含量,活性为天然酶的89.1％。缺辅基蛋白,重组酶与天然酶都是由二个相同的亚基组成;重组酶的吸收光谱与荧光光谱与天然酶几乎一样,而缺辅基蛋白则有较大的差异;从园二色谱的分析得知,缺辅基蛋白不含有α—螺旋,而天然酶和重组酶中α螺旋的含量分别为21％和20％;缺辅基蛋白比天然酶或重组酶具有更大的巯基反应性。     

The dual role of thrombin's anion-binding exosite-I in the recognition and cleavage of the protease-activated receptor 1.

The role of thrombin anion-binding exosite-I in the recognition and cleavage of the extracellular domain of the seven transmembrane domain thrombin receptor (PAR1) was determined using site-directed mutagenesis. Basic residues in anion-binding exosite-I (Arg35, Arg36, Arg67, Arg73, Arg75, Arg77A, Lys81, Lys109, Lys110 and Lys149E) were substituted with glutamines and the resultant recombinant mutant thrombins were used to determine kinetic parameters for the cleavage of a peptide (PAR38-60) based on the PAR1 extracellular domain. Compared with wild-type thrombin, replacement of Arg67 and Arg73 had a dramatic effect on the cleavage of PAR38-60 (k(cat)/K(m) = 1.8 x 10(6) and 4.6 x 10(6) vs 9.2 x 10(7) M(-1).s(-1)), whereas the remaining mutations of the anion-binding exosite-I of thrombin had a less pronounced effect, with k(cat)/K(m) values ranging from 3.3 x 10(7) M(-1). s(-1) (R77(a)Q) to 5.8 x 10(7) M(-1).s(-1) (K109Q). The ability of thrombin mutants to activate platelets paralleled that of PAR38-60 cleavage, whereas their ability to clot fibrinogen differed profoundly, as did their susceptibility to hirudin inhibition. Results are interpreted with respect to known interactions of thrombin with thrombomodulin, hirudin, rhodniin and heparin cofactor II. We conclude that the basic residues of anion-binding exosite-I contribute significantly to enhancing the rate of complex formation in two ways; the first (general) ensures electrostatic steering of ligands with complementary electrostatic fields, the second (specific) involves a combination of molecular contacts within the complex that is unique for each ligand.