Changes in structure of alpha 2-macroglobulin upon reaction with trypsin as assessed by light scattering and differential scanning calorimetry.

Employing a combination of static and dynamic light scattering, as well as differential scanning calorimetry (DSC), the structural changes which appear in alpha 2-macroglobulin (alpha 2M) upon trypsin binding have been further characterized. Light-scattering measurements suggest that a 15% reduction in both the hydrodynamic radius and radius of gyration occurs when two molecules of trypsin complex to alpha 2M. Approx. 85% of this trypsin-induced compaction results from the binding of the first proteinase. A complementary result was obtained from DSC measurements in which the major fraction of the trypsin-induced conversion of alpha 2M to a single more thermally stable form results from interaction with the first proteinase molecule. These observations support a functionally asymmetric model of trypsin binding to alpha 2M in which the significant reduction in size of the complex is primarily due to the initial interaction of alpha 2M with a single proteinase molecule.

Immunologic and physicochemical evidence for conformational changes occurring on conversion of human mast cell tryptase from active tetramer to inactive monomer. Production of monoclonal antibodies recognizing active tryptase.

The catalytic activity of human tryptase, a mast cell neutral endoprotease, is expressed when the enzyme is in its tetrameric form, but is lost under physiologic conditions concomitant with a quaternary structural alteration involving conversion to a monomeric form. The associated changes in the CD spectra noted in the current study indicate accompanying alterations in the secondary structure of the protein. In particular, the progressive disappearance of the negative minimum centered at 228 nm suggests an effect on beta-sheet structure, which may be important for monomer-monomer interaction and/or stabilization of catalytic activity. Dextran sulfate, like heparin, stabilizes the catalytic activity and quaternary structure of tryptase and also maintains the native secondary structure of the enzyme at and beyond a temperature of 40 degrees C. Dextran sulfate-stabilized tryptase therefore was used as an immunogen to which were produced three murine mAb (B2, C11, and G4) recognizing the catalytically active form of the enzyme. Inactive tryptase bound to plastic microtiter wells was not recognized by any of the newly made antibodies, whereas inactive tryptase in solution was recognized by G4, which when biotinylated, could be used as a detector antibody in a sandwich ELISA for tryptase. Each of the newly made mAb recognized the catalytically active form of tryptase. Thus, alterations in epitopes, perhaps reflecting tertiary structural alterations as well as changes in secondary and quaternary conformations, occur with tryptase inactivation. A pragmatic result of these newly generated antibodies is the affinity purification to homogeneity of active tryptase by sequential chromatography with B2 coupled to CH-Sepharose and heparin-agarose. Tryptase purified by this technique had a specific activity with p-tosyl-L-arginine methyl ester of 117 +/- 9 U/mg and had 3.9 +/- 0.3 active sites per molecule of active enzyme (134,000 m.w.) as titrated with p-nitrophenyl-p'-guanidinobenzoate. The spectral and immunologic data in the current study are consistent with concerted conformational alterations in the secondary and tertiary as well as quaternary structures of tryptase associated with loss of catalytic activity. Failure to reverse any of these alterations with dextran sulfate suggests that the pathway of tetramer assembly in vivo is more complicated than simple subunit association.

A hybrid Escherichia coli alkaline phosphatase formed on proteolysis.

Cleavage of bacterial alkaline phosphatase by trypsin at the R-11, A-12 bond of both subunits results in changes in the structure and function of the enzyme as previously reported (Roberts, C. H., and Chlebowski, J. F. (1984) J. Biol. Chem. 259, 729-733; Roberts, C. H., and Chlebowski, J. F. (1985) J. Biol. Chem. 260, 7557-7561). A hybrid dimer has been formed by cleaving the R-11, A-12 bond of only one of the two subunits. This enzyme species has been purified and characterized to investigate subunit interactions of this hybrid dimeric enzyme species. Subunit interactions were observed using various methods to study functional and structural properties of the enzyme. In a kinetic study the T-2/A-12 hybrid enzyme was found to have a Vmax similar to the A-12 fully trypsin-modified enzyme. On exposure to EDTA the hybrid was found to lose activity at essentially the same rate as the A-12 enzyme presumably as a consequence of loss of metal ions required for function. On adding metal ions back to the apoenzyme form, activity of the hybrid was reconstituted to a degree similar to that of the native enzyme whereas the activity of the A-12 enzyme was reconstituted to a much lesser extent. The Tm of the hybrid measured by differential scanning calorimetry was closer to the value obtained for the A-12 enzyme than the T-2 enzyme but circular dichroic spectra indicated secondary structural features of the hybrid different from both symmetrical forms of the enzyme. These results provide evidence for strong subunit interactions in the alkaline phosphatase dimer.

Proteolytic modification of Escherichia coli alkaline phosphatase.

Proteolytic modification of the native alkaline phosphatase dimer is restricted to sites in the amino-terminal portion of the sequence. Complementing previous studies of the product of trypsin cleavage at the R-11, A-12 bond (Roberts, C. H., and Chlebowski, J. F. (1984) J. Biol. Chem. 259, 729-733; Roberts, C. H., and Chlebowski, J. F. (1984) J. Biol. Chem. 260, 7557-7561) circular dichroic spectroscopy indicates that cleavage at this site results in a rearrangement of secondary structure and change in tertiary structure as monitored in the far and near UV regions, respectively. Under more vigorous reaction conditions, trypsin cleaves at the R-35, D-36 bond. The deletion of an additional 24 residues yields a species whose functional and structural properties are similar to the initial product of trypsin cleavage. Treatment of the enzyme with Protease V-8 results in cleavage at the E-9, N-10 bond. In contrast to the products of trypsin treatment, this truncated enzyme is similar to the native enzyme. These results indicate that the residues at the N-10 and R-11 positions play a unique role in maintaining the structural integrity and catalytic potency of the enzyme although this locus is distant from the enzyme active centers. These observations are discussed in terms of the three-dimensional structure of the enzyme.

Crystals of a trypsin-modified alkaline phosphatase. Preliminary crystallographic characterization.

Trypsin-modified alkaline phosphatase from Escherichia coli has been crystallized in a form distinct from the two known crystal forms of the native enzyme. The large well diffracting crystals belong to the orthorhombic space group P2(1)2(1)2(1), possess unit cell dimensions a = 56.0 A, b = 136.0 A, c = 283.9 A with 2 dimers per asymmetric unit, and are suitable for high resolution x-ray crystallographic studies. The observed structural and functional differences between the native and modified molecules are a result of peptide bond cleavage at Arg10-Ala11 with loss of the NH2-terminal decapeptide in both subunits of the dimer.

3-13C-methionine-labelled E. coli alkaline phosphatase.

3-13C-methionine has been biosynthetically incorporated into E. coli alkaline phosphatase using strain CW3747 which is auxotrophic for Met. 13C NMR of the dimeric native enzyme labelled at the eight methionine residues of the primary structure shows a dispersion of resonance signals permitting resolution of at least five methionine environments, none of which coincide with the chemical shift position of free methionine. At acid pH, 13C signal intensity is shifted to a chemical shift consistent with solvent exposure. However, three discrete resonances are observed, suggesting a retention of defined structure. The labelled protein thus can serve as a probe of conformational alterations of the enzyme.

Isolation of active subforms of alpha 2-macroglobulin.

Anion-exchange chromatography is shown to permit resolution and separation of subforms of the serum glycoprotein alpha 2-macroglobulin. The subforms differ dramatically in their stability as judged by differential scanning calorimetry, undergoing thermally induced unfolding at temperatures of 61 and 69 degrees C respectively. In addition, the proteinase-binding stoichiometry of the subforms differs by a factor of 2, with the more- and less-stable forms binding 2 and 1 mol of proteinase per mol of tetramer respectively. The calorimetric stability of the two forms is differentially affected on treatment with neuraminidase, suggesting that the nature of glycosylation may in part account for the observed differences in physical and functional properties.

Trypsin-modified alkaline phosphatase. Formation of apoenzyme monomer and hybrid dimer.

The cleavage of an amino-terminal decapeptide from Escherichia coli alkaline phosphatase has been previously described (Roberts, C. H., and Chlebowski, J. F. (1984) J. Biol. Chem. 259, 729-733) by this laboratory. The modest reduction in specific activity of the modified enzyme is paralleled by an apparent alteration in the Zn(II) affinity at one of the three active center metal ion binding sites. In contrast to the behavior of the native enzyme, formation of the metal-free apoprotein results in an irreversible loss of catalytic activity; phosphohydrolase activity is not restored on addition of Zn(II) and Mg(II). Differential scanning calorimetry and velocity sedimentation data indicate that the apo form of the modified enzyme exists as a monomer form which, while capable of binding Zn(II) does not readily reassociate to active dimer. Processive cleavage of the amino termini of the dimer by trypsin results in the transient formation of a hybrid dimer consisting of cleaved and uncleaved subunits. This species can be directly observed and isolated by taking advantage of the differential chromatographic mobility of the native "isozymes" and the resulting products. Coupled with improved procedures for the preparation of the modified protein, these data indicate that the amino-terminal modification results in alterations in the subunit interface domain and provides a species (the hybrid dimer) for the investigation of the propagation of these effects.

Naturally-occurring pituitary growth hormone is phosphorylated.

The results of this communication show that ovine growth hormone (oGH) contains organically-bound phosphorous. The phosphorous content of growth hormone, lot S-11, is 1:3 (mol/mol) and that of lot S-12 is 1:6 (mol/mol). Results of 31P NMR studies suggest that the phosphorous exists in two chemical forms: as a monophosphoryl ester and as a phosphodiester. Evidence is provided which demonstrates that growth hormone can be phosphorylated in vitro with the catalytic subunit of protein kinase.

Differential scanning calorimetry of Cd(II) alkaline phosphatases.

Differential scanning calorimetry has been employed to monitor structural alterations induced in the dimeric enzyme alkaline phosphatase on binding of Cd(II) (to the metal-free apoenzyme) and phosphate (Pi) (to the Cd(II) enzyme). Cd(II) addition to the apoenzyme at pH 6.5 results in an increased transition temperature, suggesting a stabilizing effect of the bound metal ion. Two distinct structural forms of the protein are detected as discrete calorimetric transitions (Tm = 69-84 degrees C; 87-94 degrees C, respectively). Distribution of the enzyme between these forms is found to depend on the exogenous Cd(II) concentration and the protocol of Cd(II) addition. These results indicate that conversion between the conformational forms is a slow process which appears to require specific levels of metal ion site occupancy. These studies, in which the exogenous Cd(II) concentration was varied from 10(-5) M to 10(-3) M suggest a structural basis for previously observed hysteretic phenomena observed on Cd(II) binding to the enzyme. Even at a minimum stoichiometry of Cd(II) (2 eq/mol of dimer) a single equivalent of Pi is sufficient to accelerate assumption of a stabilized form of the protein (Tm = 90 degrees C). This is followed by a slow structural change paralleling the time course of formation of the functional 2 Cd(II) phosphoryl enzyme which displays two calorimetric transitions (Tm = 65 degrees C, 88 degrees C). The low temperature transition does not appear if Pi is initially present at millimolar concentrations and is abolished on addition of Pi at concentrations in excess of 0.1 mM. These observations suggest the presence of a second, distinct Pi binding site on the 2 Cd(II) phosphoryl enzyme. This is supported by the changes observed in the 31P NMR chemical shift of Pi added to comparable enzyme samples. These data, including assessment of the effect of the presence of Mg(II), are discussed in terms of the mechanism of metal ion association to the enzyme and rearrangement of bound metal ions induced by Pi binding.

Trypsin modification of Escherichia coli alkaline phosphatase.

A trypsin-modified form of Escherichia coli alkaline phosphatase has been isolated, purified, and characterized. The native enzyme, previously thought to be resistant to proteases, shows a loss of 20% of its activity after a 30-min exposure to 10% trypsin. No further loss is seen after 3 h; this is in contrast to the apoenzyme which loses essentially all restorable activity (addition of saturating Zn(II) and Mg(II) restores activity to the apoenzyme) when exposed to trypsin. Under these conditions, a single major peptide is produced, cleaved at the Arg-10 Ala-11 bond, which is purified using a chromatographic technique that separates proteins according to their pI (chromatofocusing). This modified alkaline phosphatase has a Vmax of 2000 mumol/h/mg (1 M Tris, pH 8.0, 20 degrees C, 1 mM p-nitrophenolphosphate) which is 22% less than the Vmax for the native enzyme. The Km for p-nitrophenolphosphate is lower for trypsin-modified alkaline phosphatase than for the native enzyme, 1.9 X 10(-5) and 4 X 10(-5) M, respectively. The KI for Pi for the native enzyme is 1.5 X 10(-5) M and for trypsin-modified alkaline phosphatase is 1 X 10(-5) M, suggesting that the reduction in Vmax is due to a reduction in the rate constant for Pi dissociation. Differential scanning calorimetry results indicate differences in the stabilities of the two species. The trypsin-modified alkaline phosphatase has a Tm of 90 degrees C which is lower than that for the reconstituted apoenzyme (93.5 degrees C) or for the native enzyme (98.5 degrees C). This modified form of alkaline phosphatase may prove to be valuable in studies concerning subunit interactions in this system as the deleted decapeptide occurs at the subunit interface region in the native structure.

Action of cathepsin D on fructose-1,6-bisphosphate aldolase.

Cathepsin D inactivated aldolase at pH values between 4.2 and 5.2; the chloride, sulphate or iodide, but not citrate or acetate, salts of sodium or potassium accelerated the rate of inactivation. Cathepsin D cleaved numerous peptide bonds in the C-terminus of aldolase, but the major site of cleavage in this region was Leu354-Phe355. The most prominent peptide products of hydrolysis were Phe-Ile-Ser-Asn-His-Ala-Tyr and Phe-Ile-Ser-Asn-His. Up to 20 amino acids were removed from the C-terminus of aldolase, but no further degradation of native aldolase was observed. By contrast, extensive degradation of the 40 000-Mr subunit was observed after aldolase was denatured. The cathepsin D-inactivated aldolase cross-reacted with antibodies prepared against native aldolase and had the same thermodynamic stability as native aldolase, demonstrated by differential scanning calorimetry and fluorescence quenching of tryptophan residues. Furthermore, the cathepsin-modified and native forms of aldolase were both resistant to extensive proteolysis by other purified cellular proteinases and lysosomal extracts at pH values of 4.8-8.0.

Differential scanning calorimetry of alpha 2-macroglobulin and alpha 2-macroglobulin-proteinase complexes.

Differential scanning calorimetry is shown to detect substantial structural alterations occurring on the association of proteinases with the serum glycoprotein alpha 2-macroglobulin. At pH 7.5, the thermally induced unfolding of the macroglobulin occurs at approx. 60 degrees C with a transition enthalpy of 17 J/g. Association of active thermolysin, trypsin and papain shifts the transition temperature to 77 degrees C (transition enthalpy 5 J/g), indicating that a substantial conformational change accompanies the binding event. The stoicheiometry of the thermolysin--alpha 2-macroglobulin association producing this change appears to be unity, implying the presence of co-operative subunit interactions in the mechanism of association. The calorimetric method provides a novel approach for the evaluation of conformational variants induced on protein-protein association or pre-existing in the purified macroglobulin.

65Zn(II), 115mCd(II), 60Co(II), and mg(II) binding to alkaline phosphatase of Escherichia coli. Structural and functional effects.

Zn(II), Cd(II), Co(II) and Mg(II) binding to apoalkaline phosphatase of Escherichia coli and the relative stabilities of the resulting metalloenzyme complexes have been measured by equilibrium dialysis and metal exchange reactions using gamma-emitting isotopes of these metals. At millimolar concentrations of these metal ions the alkaline phosphatase dimer binds three pairs of metal ions (A, B, and C sites). One of these pairs dialyzes readily without detectable change in the structure or function of the enzyme (C site). Of the remaining two pairs, the binding affinity of both for Zn(II) and Cd(II) is increased by formation of the phosphoenzyme intermediates. Cd(II) is bound less tightly to both A and B sites than Zn(II), and at pH 6.5 Cd(II) is induced to bind to the B sites by formation of the phosphate complexes. Mg(II), 5-10 mM, competes successfully with the IIB metal ions for the second or lower affinity pair of binding sites (B sites), although Mg(II) is a relatively poor competitor on an equimolar basis, especially for Cd(II). Binding of metal ions to the apoenzyme appears to be a cooperative process involving conformational changes in the protein which are not readily reversible. The initial binding of a pair of Zn(II) or Cd(II) ions to the apoenzyme is characterized by equilibrium constants of 10(-5) to 10(-7) M for Zn(II) and 10(-4) to 10(-5) M for Cd(II). Following the cooperative binding of all three pairs of metal ions, however, re-establishment of equilibrium by dialysis indicates binding constants of less than 10(-8) M for Zn(II) and less than 10(-6) M for Cd(II) at the sites of greatest affinity (A sites). Binding of Mg(II) or Cd(II) to the B site, once the A site is occupied, increases the phosphorylation rate of the Cd(II) enzyme by 20-fold. In the presence of saturating concentrations of Mg(II) complete activity is restored to the apoenzyme by 2 Zn(II) ions. In the absence of Mg(II) as many as 6 Zn(II) ions may be required before complete restoration is achieved. Roles for the A and B site metal ions in the catalytic mechanism are discussed.

Serine hydroxymethyltransferase. 31P nuclear magnetic resonance study of the enzyme-bound pyridoxal 5'-phosphate.

The environment of the phosphate group of pyridoxal-P bound at the active site of cytosolic serine hydroxymethyltransferase has been investigated by 31P NMR spectroscopy. In the holoenzyme, the pyridoxal-P chemical shift is pH-dependent with a pKa of 6.45. The chemical shift of the bound pyridoxal-P is shifted upfield about 0.3 ppm from the signal for free pyridoxal-P. Saturation of the active site with the substrates L-serine, glycine, and tetrahydrofolate does not alter the chemical shift or the pKa of the phosphate group. The addition of these substrates does, however, alter the absorption and circular dichroism spectra of the bound coenzyme, reflecting environmental changes of the pyridine ring-Schiff's base system. We conclude from these studies that the phosphate group of the bound coenzyme is exposed to the solvent. The reorientation and conformational changes of the pyridoxal-P ring which take place during the formation of enzyme-substrate complexes do not appear to change the environment of the phosphate moiety of the coenzyme.

Differential scanning calorimetry of cytoplasmic aspartate transaminase.

Differential scanning calorimetry has been applied to study factors affecting the thermally induced denaturation of cytoplasmic aspartate aminotransferase, a dimeric pyridoxal enzyme. The consequences of binding of coenzyme and substrate derivatives to both the apo and holo forms of the enzyme were investigated and are interpreted in terms of the stabilization of the native form of the enzyme. The binding of pyridoxal phosphate coenzyme increases the thermal stability of the apoenzyme by approximately 27 kcal mol-1 as judged by the change in free energy differences between the native and denatured states of the protein. The stabilization produced by coenzyme binding to the apoprotein appears to be primarily due to the Schiff's base and phosphoryl moieties of the coenzyme; association of the pyridine ring component is without significant structural consequence. Pyridoxal phosphate binding to the subunits of the dimer occurs in a noncooperative fashion as judged by the appearance of transitions unique to the apo, holo, and intermediate enzyme forms in a calorimetric titration. Holoenzyme stability depends on the chemical nature of the catalytically significant group occupying the C-4' position of the bound coenzyme. The stabilization afforded by binding of the aldehyde form (pyridoxal phosphate) which exists as an internal Schiff's base with Lys 258 is diminished when this bond is chemically reduced or when the aldehyde is replaced by an amine (pyridoxamine phosphate). Apoenzyme is also shown to be stabilized by the presence of substrates in the absence of coenzyme. The differential scanning calorimetry results thus confirm previous findings derived from nuclear magnetic resonance studies on the ability of apoenzyme to bind substrates (Martinez-Carrion, M. Cheng, S., and Relimpio, A. (1973) J. Biol. Chem. 248, 2153-2160). Substrates and their analogues perturb the holoenzyme stability and the order of increasing influence on the pyridoxal form of the holoenzyme is aspartate, erythro-hydroxyaspartate, alpha-ketoglutarate, and alpha-methylaspartate. While all these compounds form stable binary enzyme-substrate complexes (Jenkins, W.T., and D'Ari, L. (1966) J. Biol. Chem. 541, 5667-5674), the complex with alpha-methylaspartate produces anomalous changes in the protein structure which are reflected in the calorimetric parameters. This suggests that caution be exercised in the use of analogues as substrate substitutes in crystallographic work. Differential scanning calorimetry also appears as a sensitive method with which to study the stereochemical dependence of ligand binding on enzyme-induced thermal stabilization. This is illustrated by the use of 4-carbon dicarboxylic acids where only those in the conformation favorable for binding are effective in stabilizing the holoenzyme.