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U Christensen

Publications and source records attributed to U Christensen.

At least 37 records · Page 2Linked to original sources
PubMed1994

Effects of pH on carboxypeptidase-Y-catalyzed hydrolysis and aminolysis reactions.

The pH dependencies of serine carboxypeptidase-Y-catalysed hydrolysis and aminolysis reactions using L-amino acids and L-amino acid amides as nucleophiles, have been studied and analyzed. The results reveal two catalytically important ionizing groups of the enzyme with rather similar pK values (5-6), the active site His397 and a possibly Glu residue, which is not only important in interactions with carboxylic groups of substrates and nucleophiles [Liao, D.-I., Breddam, K., Sweet, R. M., Bullock, T. & Remmington, S. J. (1992) Biochemistry 31, 9796-9812], but also indirectly play a role in catalysis. This explains the pH behaviour of hydrolysis of both peptide and ester substrates and further, that L-amino acid amides are better nucleophiles in aminolysis reactions than L-amino acids.

Amino Acids↗
PubMed1994

Effects of introduced aspartic and glutamic acid residues on the P'1 substrate specificity, pH dependence and stability of carboxypeptidase Y.

Carboxypeptidase Y is a serine carboxypeptidase isolated from Saccharomyces cerevisiae with a preference for C-terminal hydrophobic amino acid residues. In order to alter the inherent substrate specificity of CPD-Y into one for basic amino acid residues in P'1, we have introduced Asp and/or Glu residues at a number of selected positions within the S'1 binding site. The effects of these substitutions on the substrate specificity, pH dependence and protein stability have been evaluated. The results presented here demonstrate that it is possible to obtain significant changes in the substrate preference by introducing charged amino acids into the framework provided by an enzyme with a quite different specificity. The introduced acidic amino acid residues provide a marked pH dependence of the (kcat/Km)FA-A-R-OH/(kcat/Km)FA-A-L-OH ratio. The change in stability upon introduction of Asp/Glu residues can be correlated to the difference in the mean buried surface area between the substituted and the substituting amino acid. Thus, the effects of acidic amino acid residues on the protein stability depend upon whether the introduced amino acid protrudes from the solvent accessible surface as defined by the surrounding residues in the wild type enzyme or is submerged below.

Amino Acid Sequence↗
PubMed1993

Reaction mechanisms of Trp120-->Phe and wild-type glucoamylases from Aspergillus niger. Interactions with maltooligodextrins and acarbose.

Interactions of wild-type and Trp120-->Phe glucoamylase with maltooligodextrin (Gx) substrates and the tight-binding inhibitor acarbose (A) were investigated here using stopped-flow fluorescence spectroscopy and steady-state kinetic measurements. All wild-type and Trp120-->Phe glucoamylase reactions followed the three-step model E + Gx(or A) (k1) <==> (k-1) EGx (or A) (k2) <==> (k-2) E*Gx(or A) (k3) --> E + P or E-A, previously shown to account for the glucoamylase-maltose system [Olsen, K., Svensson, B., & Christensen, U. (1992) Eur. J. Biochem. 209, 777-784]. K1 = k-1/k1, k2, and k-2, and the catalytic constant, k3, are determined. Binding of maltooligodextrins in the first reaction step is weak, with little difference between wild-type and Trp120-->Phe glucoamylase. The second step, involving a conformational change, in contrast, is strongly influenced by the mutation and by the substrate length. Here wild-type glucoamylase reacts faster and forms more stable intermediates the longer the substrate. In contrast, Trp120-->Phe reacts slower the longer the substrate. The effect of the mutation is thus smallest on maltose. The Trp120-->Phe substitution reduces the fluorescence signal only by 12-20%, indicating that other tryptophanyl residues are important in reporting the conformational change. Trp120 also strongly influences the actual catalytic step, since the mutation decreases the kc values 30-80-fold. Acarbose behaves similar to maltotetraose in the first and the second steps with wild-type but not the Trp120-->Phe glucoamylase. Also, a third step in the acarbose reaction which parallels the catalytic step is strongly affected by the mutation. The rate constant k3 increases 200-fold.

Acarbose↗
PubMed1993

Different N-terminal forms of alpha 2-plasmin inhibitor in human plasma.

Mature alpha 2-plasmin inhibitor in human plasma has 12 more N-terminal residues than hitherto anticipated. The first residue is the methionine at position 28, downstream from the N-terminus of the pre-protein. The cDNA sequence predicts that the site cleaved upon formation of the mature inhibitor is a typical signal-peptidase recognition site. The mature inhibitor (464 residues) and the previously reported, and presumably degraded, form with N-terminal asparagine (452 residues), are present in plasma in about equal amounts. They both form a stable complex with plasmin. Recent studies on a recombinant alpha 2-plasmin inhibitor suggest that the 12 additional residues have functional implications [Sumi, Ichikawa, Nakamura, Miura and Aoki (1989) J. Biochem. 106, 703-707].

Amino Acid Sequence↗
PubMed1993

Activity modulation of the fast and slow isozymes of human cytosolic low-molecular-weight acid phosphatase (ACP1) by purines.

The activity modulation of homogeneous isozymes of the human cytosolic M(r) 18,000 acid phosphatase (ACP1) by purines has been investigated. A pronounced difference in the response of fast and slow isozymes of the same genetic type was observed, while identical properties were found for fast isozymes encoded by different alleles (ACP1 X A, B and C), as well as for the corresponding slow isozymes. The catalytic rate constant (kc) of the fast isozymes was increased 5.1-fold by hypoxanthine and decreased 40% by adenine, while the kc of the slow isozymes was unaffected by hypoxanthine but increased 4.6-fold by adenine. This finding and the genetically-determined differences in the relative quantities of the fast and slow isozymes account for the well-known phenotypic differences in activity modulation. The kinetic results strongly indicate that the effector binds to the free enzyme, as well as to the enzyme-substrate complex. Activating effectors showed a higher affinity for the free enzyme than for the enzyme-substrate complex, while the reverse was true with the inhibitor. The results exclude the possibility that effector and substrate bind to the same site of the enzyme; parasteric binding to adjacent sites is suggested.

Acid Phosphatase↗
PubMed1992

Mechanism of carboxypeptidase-Y-catalysed peptide semisynthesis.

The initial rate steady-state kinetics of carboxypeptidase-Y-catalyzed hydrolysis and aminolysis reactions with some alpha-N-benzoyl-L-tyrosinyl compounds has been investigated using L-valinamide as the nucleophile in aminolysis. Hydrolysis of alpha-N-benzoyl-L-tyrosinyl ethyl ester, 4-nitroanilide, and -amide has been studied in the pH range 4-9. The results are interpreted in terms of the classical serine proteinase mechanism, which involves enzyme-substrate complex formation, followed by acylation and deacylation of the enzyme. The three reactions share the same deacylation step. It is rate-determining with the ester substrate, but with the 4-nitroaniline acylation is and this is even more pronounced with the amide. From the pH dependencies, no change of rate-determining step is apparent in the range pH 4-9. For the 4-nitroanilide and the amide substrates, the kinetic parameter, Kc/Km, is influenced by an ionizing group with a pK value of 6. Probably this is the active-site histidine residue, which thus is active in acylation in its deprotonated form. That group affects the deacylation reaction similarly as seen from the kinetics of the ester substrate. Aminolysis occurs in parallel to hydrolysis in the presence of reactive nucleophiles. Here L-valinamide was used as model nucleophile. The analysis of the observed kinetic effects of L-valinamide on the initial rate behaviour of carboxypeptidase-Y-catalyzed hydrolysis reactions suggests a reaction mechanism which involves (a) the binding of the free nucleophile to the free enzyme and (b) reaction of the free nucleophile with the acyl-enzyme complex forming an enzyme-aminolysis product complex, which dissociates into the free enzyme and the aminolysis product. The reactions are characterized by a number of kinetic parameters, the values of which are determined. The results of aminolysis progress reactions indicate that the formation of the product in high yields is strongly dependent on the leaving group of the substrate. The initial production of aminolysis product, however, is the same for the three substrates. But the fact that their Kc/Km values differ by several orders of magnitude leads to significantly different progresses of the aminolysis. The ester substrate is the only one that efficiently competes with and hinders the hydrolysis of the aminolysis product.

Acylation↗
PubMed1992

Stopped-flow fluorescence and steady-state kinetic studies of ligand-binding reactions of glucoamylase from Aspergillus niger.

The presteady-state and steady-state kinetics of the binding and hydrolysis of substrates, maltose and isomaltose, and the transition-state analogue, gluconolactone, by glucoamylase from Aspergillus niger were investigated using initial-rate, stopped-flow and steady-state methods. The change in the intrinsic fluorescence of the enzyme was monitored. Distinct mechanistic differences were observed in the interaction of the enzyme with maltose compared to isomaltose. Hydrolysis of maltose requires a three-step mechanism, whereas that of isomaltose involves at least one additional step. The rates of an observed conformational change, which is the second discernible step of the reactions, clearly show a tighter binding of maltose compared to isomaltose, probably because the reverse rate constants differ. Compared to the non-enzymic hydrolysis the transition-state stabilization energy of glucoamylase is approximately -66 kJ/mol with maltose and only -14 kJ/mol with isomaltose. Kinetic analysis of the binding of the inhibitor, gluconolactone, implies that independent interactions of two molecules occur. One of these, apparently, is a simple, fast association reaction in which gluconolactone is weakly bound. The other resembles binding of maltose, involving a fast association followed by a conformational change. Based on the results obtained, we propose new reaction mechanisms for Aspergillus glucoamylase.

Aspergillus niger↗
PubMed1992

Interaction between bovine trypsin and a synthetic peptide containing 28 residues of the bait region of human alpha 2-macroglobulin.

The time course of the interaction between trypsin and a synthetic peptide corresponding to a segment (residues 676-703) of the bait region (residues 666-706) of human alpha 2-macroglobulin (alpha 2M) was studied by measuring the generation of cleavage products as a function of time by HPLC. Three primary cleavage sites for trypsin were present in the synthetic peptide. The fastest cleavage occurred at the bond corresponding to Arg696-Leu in alpha 2M with an estimated kcat/Km = 1-2 x 10(6) M-1.s-1. This value is of the same magnitude as that characterizing the interaction of alpha 2M and trypsin when taking into account the fact that alpha 2M is a tetramer, kcat/Km = 5 x 10(6) M-1.s-1 [Christensen, U. & Sottrup-Jensen, L. (1984) Biochemistry 23, 6619-6626]. The values of kcat/Km for cleavage at bonds corresponding to Arg681-Val and Arg692-Gly in alpha 2M were 1.5 x 10(5) M-1.s-1 and 1.3 x 10(5) M-1.s-1, respectively. Cleavage of intermediate product peptides was slower, with kcat/Km in the range 13-1.3 x 10(6) M-1.s-1. The value of Km determined for fast cleavage in the synthetic peptide was 8-10 microM. 1H-NMR spectroscopy indicated no ordered structure of the peptide. Hence, the very fast cleavage of the peptide is compatible with a loose structure that readily adopts a conformation favorable for recognition and cleavage by trypsin.

Amino Acid Sequence↗
PubMed1992

Two different Ca2+ ion binding sites in factor VIIa and in des(1-38) factor VIIa.

The Ca2+ ion binding of factor VIIa and the derivative lacking the gamma-carboxyglutamic acid domain, des(1-38) factor VIIa, was investigated using intrinsic protein fluorescence and Tb3+ ion phosphorescence methods. Binding of Ca2+ ions giving rise to a decrease in the intrinsic protein fluorescence (approximately 50% at saturating conditions) is seen with both proteins. Each of the saturation curves is in accordance with the formation of a 1:1 complex of factor VIIa-Ca2+ (KD approximately 30 microM) and des(1-38) factor VIIa-Ca2+ (KD approximately 40 microM)). Yet another Ca2+ ion binding site reveals itself in each protein in Tb3+ ion phosphorescence experiments. Ca2+ ion competition studies have showed 1:1 complexes (KD's approximately 2 mM). The results are interpreted in terms of two different Ca2+ ion binding sites, one in the EGF-1 domain and one in the Gly-209-Gln-221 loop of the serine proteinase part.

Animals↗
PubMed1992

Positive co-operative binding at two weak lysine-binding sites governs the Glu-plasminogen conformational change.

The kinetics of a series of Glu-plasminogen ligand-binding processes were investigated at pH 7.8 and 25 degrees C (in 0.1 M-NaCl). The ligands include compounds analogous to C-terminal lysine residues and to normal lysine residues. Changes of the Glu-plasminogen protein fluorescence were measured in a stopped-flow instrument as a function of time after rapid mixing of Glu-plasminogen and ligand at various concentrations. Large positive fluorescence changes (approximately 10%) accompany the ligand-induced conformational changes of Glu-plasminogen resulting from binding at weak lysine-binding sites. Detailed studies of the concentration-dependencies of the equilibrium signals and the rate constants of the process induced by various ligands showed the conformational change to involve two sites in a concerted positive co-operative process with three steps: (i) binding of a ligand at a very weak lysine-binding site that preferentially, but not exclusively, binds C-terminal-type lysine ligands, (ii) the rate-determining actual-conformational-change step and (iii) binding of one more lysine ligand at a second weak lysine-binding site that then binds the ligand more tightly. Further, totally independent initial small negative fluorescence changes (approximately 2-4%) corresponding to binding at the strong lysine-binding site of kringle 1 [Sottrup-Jensen, Claeys, Zajdel, Petersen & Magnusson (1978) Prog. Chem. Fibrinolysis Thrombolysis 3, 191-209] were observed for the C-terminal-type ligands. The finding that the conformational change in Glu-plasminogen involves two weak lysine-binding sites indicates that the effect cannot be assigned to any single kringle and that the problem of whether kringle 4 or kringle 5 is responsible for the process resolves itself. Probably kringle 4 and 5 are both participating. The involvement of two lysine binding-sites further makes the high specificity of Glu-plasminogen effectors more conceivable.

Aminocaproic Acid↗
PubMed1992

Kinetics and mechanism of proteinase-binding of pregnancy zone protein (PZP). Appearance of sulfhydryl groups in reactions with proteinases.

Proteinase binding by pregnancy zone protein (PZP), an alpha-macroglobulin involves bait region cleavages, association of dimeric-PZP into tetrameric and reaction of internal gamma-glutamyl-beta-cysteinyl thiol esters of PZP with proteinase side chains. The product is an equimolar enzyme-PZP(tetramer) covalently linked complex with four free sulfhydryl groups. The kinetics of the appearances of sulfhydryl groups during the reaction of PZP with chymotrypsin has been investigated using stopped-flow and conventional mixing techniques over a broad concentration range. Thiol ester cleavages followed double exponential decays corresponding with two steps. The faster one resulted in the appearance of three sulfhydryl groups with an observed rate constant, k(obs) = k1.1 + k1.2 delta E, dependent on the excess concentration of chymotrypsin, delta E, and k1.1 = 0.03 s-1 and k1.2 = 4 x 10(4) M-1 s-1. The last sulfhydryl group appeared in a slower step, with similar concentration dependence and k2.1 approximately 0.003 s-1 and k2.2 approximately 5 x 10(3) M-1s-1. Covalent binding of the enzyme apparently was simultaneous with the faster thiol ester cleavage step. Based on these and previous results a model of the reaction mechanism of the proteinase binding reaction of PZP is proposed. It consists of four major steps: (i) Bait region cleavage of PZP-dimers by the enzyme, (ii) fast association of enzyme-PZP(dimer) species with native PZP or with another enzyme-PZP(dimer) compound resulting in release of one of the associated enzyme molecules (iii) reaction of an average of three thiol esters of the enzyme-PZP(tetramer) intermediate with the associated internal enzyme molecule or with an external one. In this step one enzyme molecule becomes covalently linked to the PZP-(tetramer), three sulfhydryl groups appear and the enzymic activity of the bound enzyme molecule decreases to the level of that of the final complex. (iv) Hydrolysis of the last thiol ester and in the presence of excess enzyme, degradation of enzyme-PZP(tetramer) complexes and formation of fragments some of which are the size of PZP(dimer) with enzyme bound.

Amino Acid Sequence↗
PubMed1991

[Drug committee--an example of a quality circle].

A theoretical comparison was undertaken between the work of the drug committee and that of the quality circle in order to investigate whether quality circles are suited as methods for organisation of ensuring the standard of quality in Danish hospitals. On the basis of an investigation of the pharmaceutical service in hospitals, the work of the drug committees was analysed and assessed. The authors found that drug committees which worked according to quality circle principles achieved their goal: To rationalise the employment of drugs in routine treatment. The authors consider that formation of quality circles is a suitable method of ensuring quality in Danish hospitals but it would probably be of decisive significance for the result that the hospital direction assumed its responsibility for the results and for coordination of the circles.

Denmark↗
PubMed1991

Stopped-flow fluorescence kinetic studies of Glu-plasminogen. Conformational changes triggered by AH-site ligand binding.

Binding of 6-aminohexanoic acid to the AH-site, a weak lysine binding site in Glu-plasminogen, alters the conformation of the molecule. The kinetics of the binding and the accompanying conformational change are investigated at pH 7.8, 25 degrees C. Changes of intrinsic protein fluorescence were measured as a function of time after rapid mixing in a stopped-flow apparatus. The results reflect a two-step reaction mechanism: Rapid association of Glu-plasminogen and 6-aminohexanoic acid (K1 = 44 mM) followed by the conformational change (k2 = 69 s-1 and k-2 = 3 s-1) with an overall dissociation constant Kd = 2.0 mM. Thus the conformational change is rather fast, t12 = 0.01 s. Its importance for the rates of Glu-plasminogen activation reactions is discussed.

Aminocaproic Acid↗
PubMed1991

Pregnancy zone protein, a proteinase binding alpha-macroglobulin. Stopped-flow kinetic studies of its interaction with chymotrypsin.

Human pregnancy zone protein (PZP) is a major pregnancy-associated plasma protein, strongly related to alpha 2-macroglobulin (alpha 2M). The proteinase binding reaction of PZP is investigated using chymotrypsin as a model enzyme. The time-course of the interaction is studied by measuring the change in intrinsic protein fluorescence of PZP-chymotrypsin reaction mixtures as a function of time after rapid mixing in a stopped-flow apparatus. Titrations show the changes of fluorescence at equilibrium to correspond with the formation of a chymotrypsin-PZP(tetramer) species. The kinetic results show the formation of the species to take place in an overall second-order process dependent on the concentrations of chymotrypsin and of PZP(dimers), k = 5 x 10(5) M-1 x s-1. Reactions of PZP-thiol groups do not give rise to fluorescence changes. The fluorescence changes most likely reflect the formation of an intermediate with intact thiol esters. Further analysis of the kinetic results suggests that the chymotrypsin-PZP(tetramer) intermediate is formed in two reaction steps: (1) initially native PZP(dimers) are cleaved at bait regions by enzyme molecules, and that is the rate determining reaction of the fluorescence changes; (2) association with another PZP(dimer) or PZP(dimer)-chymotrypsin complex in a very fast reaction that leads to the formation of 1:1 -chymotrypsin-PZP(tetramer) intermediate, probably with intact thiol esters. The interactions studied apparently are established early in the path of the reaction and the fluorescence changes probably reflect noncovalent enzyme-PZP contacts, which are not changed when covalent binding occurs. Further, fluorescence changes are seen only in reactions of PZP with enzymes, not with methylamine.

Chymotrypsin↗
PubMed1989

Pregnancy zone protein, a proteinase-binding macroglobulin. Interactions with proteinases and methylamine.

Human pregnancy zone protein (PZP) is a major pregnancy-associated plasma protein, strongly related to alpha 2-macroglobulin (alpha 2M). Its properties and its reactions with a number of enzymes, particularly chymotrypsin, and with methylamine have been investigated. It is concluded that native PZP molecules are dimers of disulfide-bridged 180-kDa subunits and that proteinase binding results in covalent 1:1 (tetrameric)PZP-enzyme complexes. Native PZP is unstable, and storage should be avoided, but when kept unfrozen at 0 degree C most PZP preparations stay native 1-3 months. The reaction of PZP with chymotrypsin involves (i) proteolysis of bait regions, (ii) cleavage of beta-cysteinyl-gamma-glutamyl thiol ester groups, (iii) some change of the conformation and quaternary structure of PZP, and (iv) the formation of covalent 1:1 chymotrypsin-PZP(tetramer) complexes in which chymotrypsin is active but shows less activity than free chymotrypsin. The emission spectra of intrinsic fluorescence show significant differences between the PZP-chymotrypsin complex and its native components, whereas no differences are observed between methylamine-reacted PZP and native PZP. Methylamine reacts with the beta-cysteinyl-gamma-glutamyl thiol ester groups of PZP in a second-order process with k = (13.6 +/- 0.5) M-1 s-1, pH 7.6, 25 degrees C. The reaction product is PZP(dimers); no PZP(tetramers) are formed. The proteinase-binding specificity of PZP is far more restricted than that of alpha 2M. Certain chymotrypsin-like and trypsin-like enzymes are bound much less efficiently than is chymotrypsin itself.(ABSTRACT TRUNCATED AT 250 WORDS)

Chymotrypsin↗
PubMed1988

Urokinase-catalysed plasminogen activation. Effects of ligands binding to the AH-site of plasminogen.

The kinetics of activation of Lys-plasminogen (Lys-77-Asn-790) and miniplasminogen (Val-442-Asn-790) catalysed by low-molecular-weight urokinase (LMW-urokinase) was investigated in the presence and absence of ligands that bind to the AH-site of the plasminogens. 6-Aminohexanoic acid and alpha-N-acetyl-L-lysine methyl ester (AcLysMe) were used. Saturation of the AH-sites of the plasminogens result in similar, but rather small positive effects on the kinetics of activation of the two plasminogens. Michaelis constants decrease approx. 2-fold and second-order rate constants (kc/Km)Pg increase approx. 1.2-fold. Michaelis constants (KPg values) were obtained using a new approach; the values were determined from the competing effects of the plasminogens on urokinase-catalysed hydrolysis of a synthetic substrate. In the pH range 7.4-8.0, only minor alterations of the values of the kinetic parameters are observed. At 25 degrees C, values of (kc/Km)Pg are approx. 3-fold less than the value at 37 degrees C, whereas KPg is not changed. We conclude that kc/Km values are approx. 10(5) M-1.s-1 and that KPg values are approx. 40 microM of urokinase-catalysed conversions of Lys- and miniplasminogen to their respective plasmins.

Binding Sites↗
PubMed1986

The action of factor Xa on peptide p-nitroanilide substrates: substrate selectivity and examination of hydrolysis with different reaction conditions.

Kinetic parameters for the action of bovine Factor Xa (EC 3.4.21.22) on 25 commercially available peptide p-nitroanilides have been determined. The selectivity constant, kc/Km, ranges from 1.5 X 10(1) to 2 X 10(6) M-1 X s-1 for the poorest and the best substates, respectively. The best substrates for Factor Xa were identified as those with arginine in the P1 position, and glycine in the P2 position. Quantitative distinction between lysine and arginine in the P1 position and other amino acids in the P2-P4 positions of the substrate is reported from the changes in the kinetic parameters for substrates differing in only a single amino acid in these positions. Effect of NaCl and CaCl2 concentrations and temperature on the action of Factor Xa on selected substrates have been assessed. Km values for Factor Xa hydrolysis of most substrates are greater than 100 microM. Solubility of the substrates consequently restricts measurements of reaction velocities to concentrations lower than desirable for optimally determining kc. Comparison of these kinetic parameters for Factor Xa with those of thrombin (Lottenberg, R., Hall, J.A., Blinder, M., Binder, E. and Jackson, C.M. (1983) Biochim. Biophys. Acta 742,539-557) for these same substrates indicates that the greater hydrolytic efficiency of thrombin is due primarily to lower Km values.

Anilides↗