Metal-ion binding and the molecular conformational properties of alpha lactalbumin.

Mammary galactosyltransferase and alpha lactalbumin are the two protein components of lactose synthase which catalyze the transfer of galactose from UDP-gal to glucose in the presence of divalent cations. Recent studies suggest that alpha lactalbumin may have a broader function in modifying cell surface carbohydrates in cell-cell interactions and cell differentiation. Since the discovery that alpha lactalbumin, like galactosyltransferase, is a metalloprotein, there has been a great deal of interest in the metal-binding properties of this protein and how these relate to the metal-ion requirements of the lactose synthase reaction. The recent availability of an X-ray crystal structure of alpha lactalbumin has provided further impetus for establishing the molecular determinants of its biological activity. This review is directed toward critically examining and integrating our present knowledge of the properties of this protein, particularly the relationship between metal-ion binding and conformational state, and how these might relate to its biological function.

Metal ion binding to the N and A conformers of bovine alpha-lactalbumin.

The binding of Ca2+, Zn2+, Tb3+, and Mn2+ to metal-free bovine alpha-lactalbumin (apo-BLA) was studied by both analytical gel filtration using isotopic metal ions and by fluorescence titration. In the absence of other metal ions, Ca2+ binds at a single site on apo-BLA. The pH dependence of pKa for calcium binding indicates that carboxylate groups of aspartic and/or glutamic residues are coordinating groups for the metal ions and that histidine residues are most likely absent from the site. An analysis of the (Ca2+) dependence of the equilibrium constant for the N-A conformational change indicates the absence of binding of this metal ion to the A conformer. Binding of zinc occurs at two sites on apo-BLA (pKa 5.05 and 2.78). Occupancy of the higher affinity site stabilizes the A state while binding at the second site has been shown to give rise to a time-dependent conformational change leading to an "expanded A state" (Kronman, M. J. and Bratcher, S. C. (1984) J. Biol. Chem. 259, 10887-10895). There are three binding sites for Tb3+ on apo-BLA with the occupancy of the site of highest affinity leading to the N conformation. Binding of terbium at a second site reduces the affinity of binding at the first one. Binding of terbium at the third site induces a time-dependent transformation to the "expanded A state" (see above for reference). There are three binding sites for Mn2+. A quantitative resolution of the affinities for each of these sites is precluded by the dependence of binding affinity on association of the Mn2+-liganded protein. At apo-BLA concentrations of the order of 4 microM (fluorescence titration), pKa for binding at the site with highest affinity is 5.8, more than an order of magnitude higher than seen at protein concentrations approaching 1 mM (Murakami, K., and Berliner, L. J. (1983) Biochemistry 22, 3370-3374). Binding of Mn2+ to apo-BLA was also found to be time-dependent in contrast with that of Ca2+ which appeared to be instantaneous. Measurements with Ca2+, Mn2+, and apo-BLA in experiments with simultaneous mixing of components revealed little if any competition of binding, i.e. Ca2+ binding was little effected and Mn2+ binding was strongly inhibited over nearly a 2000-fold range of concentrations of the latter ion. With sequential mixing of components (pre-equilibration of protein with Mn2+), markedly increased binding of Mn2+ was observed and binding of Ca2+ at two sites was seen.

Conformational changes induced by zinc and terbium binding to native bovine alpha-lactalbumin and calcium-free alpha-lactalbumin.

Terbium at submillimolar concentrations appears to bind to the calcium site of apo bovine alpha-lactalbumin and stabilizes the N conformation (fluorescence criterion; Kronman, M. J., Sinha, S., and Brew, K. (1981) J. Biol. Chem. 256, 8582-8586). At millimolar concentrations however, it binds additionally to a low affinity site of both apo- and calcium-liganded protein, inducing a time-dependent conformational change to an "expanded A-like state." The pH dependence of the transformation implicates the alpha-amino group of glutamic acid 1 of the protein in the binding process. The Zn2+ concentration dependence of the fluorescence of the calcium-free protein indicates there to be two binding sites for this metal ion in agreement with the binding studies with Zn2+ (Bratcher, S.C., and Kronman, M. J. (1984) J. Biol. Chem. 259, 10875-10886). Binding of Zn2+ at submillimolar concentrations stabilizes the A conformation of the protein in contrast to what was observed with Tb3+ at comparable metal ion concentrations. Millimolar concentrations of Zn2+ induce a time-dependent conformational change in both calcium-free and calcium-liganded alpha-lactalbumin to produce an "expanded A-like state" comparable to that seen with terbium at similar concentrations. In contrast to the "expanded A state" induced by high concentrations of zinc or terbium, a "collapsed A state" results from binding of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer species, EDTA, and ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (Kronman, M. J., and Bratcher, S. C. (1983) J. Biol. Chem. 258, 5707-5709), lysine methyl ester, arginine methyl ester, and histidine and by increasing ionic strength of the medium. Since the Zn2+ form of the protein (A conformation) promotes lactose synthetase activity (Kronman et al. cited above), the conformational flexibility of alpha-lactalbumin is likely to be of importance in the formation of the catalytically active complex with galactosyltransferase.

An experimental artifact in the use of chelating metal ion buffers. Binding of chelators to bovine alpha-lactalbumin.

The binding of EDTA and EGTA to bovine alpha-lactalbumin was shown to stabilize the native conformation relative to that characteristic of the metal-free protein (A conformer). Fluorescence titration of the metal-free protein with Ca2+ in the presence of EDTA was markedly influenced by micromolar concentrations of EDTA such that the apparent association constant of calcium binding would be significantly larger than obtained in the absence of chelator. These observations explain the discrepancy in values of K alpha reported by different investigators for binding near neutral pH: 2.75 X 10(6) M-1 (Kronman, M. J., Sinha, S. K., and Brew, K. (1981) J. Biol. Chem. 256, 8582-8586); 6.3 X 10(8) M-1 (Permyakov, E. A., Yarmolenko, V. V., Kalinichemko, L. P., Morozova, L. A., and Burstein, E. A. (1981) Biochem. Biophys. Res. Commun. 100, 191-197); and 4 X 10(9) M-1 (Murakami, K., Andree, P., and Berliner, L. J. (1982) Biochemistry 21, 5488-5494). The last two high values were obtained by fluorescence titration in the presence of EGTA, while the former lower one was determined by a gel filtration technique in the absence of chelators. The anomolous association constants for calcium binding and the alteration of a conformational equilibrium observed in the presence of chelators demonstrate the need for great care in the use of chelating metal ion buffers in the studying of metalloproteins.

Characteristics of the binding of Ca2+ and other divalent metal ions to bovine alpha-lactalbumin.

Removal of the tightly bound Ca2+ ion from bovine alpha-lactalbumin (Hiraoka et al. (1980) Biochem. Biophys. Res. Commun. 95, 1098-1104) produces a pronounced conformational change, as indicated by fluorescence and absorbance changes. These changes closely resemble the changes that occur on acid denaturation of the native protein. The binding of ions to apo-alpha-lactalbumin at pH 7.4 has been examined by monitoring fluorescence changes and by direct binding measurements with 45CaCl2. The results indicate the presence of two Ca2+ binding sites on apo-bovine alpha-lactalbumin, a stronger binding site (Ka of 2.7 X 10(6) M-1) and a weaker site (Ka of 3.1 X 10(4) M-1); the fluorescence changes on Ca2+ rebinding correlate with saturation of the stronger site. Mn2+ can also bind to restore a "native" structure but with a lower affinity(Ka of 3.5 X 10(5) M-1). Zn2+ and Co2+ do not produce this change, but Zn2+ (1 mM) greatly inhibits the binding of 45Ca2+ in the direct binding assay and produces a time-dependent displacement of Ca2+ from the native protein to an apo-protein-like conformation. Co2+ does not produce these effects. Studies with metal-depleted galactosyltransferase activated with Zn2+ or Co2+ and apo-alpha-lactalbumin or Ca2+-saturated alpha-lactalbumin show that the Ca2+, Zn2+, and apo-alpha-lactalbumin are all able to bind with galactosyltransferase to produce an active lactose synthase complex.

The pH dependence of tryptophan fluorescence of goat alpha-lactalbumin with particular reference to the effect of binding of an impurity from milk.

Two processes giving rise to changes in tryptophan fluorescence of goat alpha-lactalbumin below pH 7 have been studied. Both of these spectral transitions are strongly influenced by the presence of an impurity which binds to the protein if its contact at acid pH with the milk mother liquor is prolonged during its preparation. Transition II, the U leads to N conversion, follows the same course at both 2 and 25 degrees C, i.e. a minimum of three abnormally titrating groups are normalized during the conformational change at both temperatures. The difference in this conclusion from that made earlier (Kronman, M.J., Jeroszko, J. and Sage, G.W. (1972) Biochim. Biophys. Acta 285, 145-166) that groups are normalized at 25 and at 2 degrees C was found to be due to binding of the impurity to the protein used in the earlier study. Transition I, which we detect between pH 7 and 4.4 and which appears to be due to perturbation of tryptophans by vicinal ionizing groups, overlaps Transition II, the U leads to N conversion. However, the fluorescence above approx. 340 nm in the emission spectrum appears to be free of contributions from the former process and can be used in a valid analysis of the thermodynamics of the latter process.

Comparative fluorescence properties of bovine, goat, human and guinea pig alpha lactalbumin. Characterization of the environments of individual tryptophan residues in partially folded conformers.

alpha Lactalbumin exists as a partially folded conformer (U form) at acid pH. A second partially folded conformer (H form) is formed above 60 degrees. Comparison of the changes in tryptophan fluorescence which occur on forming U and H for the bovine, goat, human and guinea pig proteins, as well as analysis of fluorescence properties for the bovine protein and an N bromo succinimide derivative of this protein, have made it possible to determine which tryptophan residues give rise to such changes in fluorescence, and to draw a distinction between the molecular structure of the U and H forms of the protein. Trp 28 and 109 in the native state transfer their excitation energy to trp 63 whose fluorescence is quenched by a pair of vicinal disulfide bridges. This process persists in the U form of the protein, but is absent in the H conformer. Most of the change in fluorescence seen in the N in equilibrium with U conversion is due to increase in yield of trp 28, while the changes in fluorescence occurring on formation of the H form are due to exposure of trp 63 and elimination of its quenching and/or excited state transfer from 28 to 109.

Chemical modification of tyrosyl and lysyl residues in goat alpha lactablbumin and the effect on the interaction with the galactosyl transferase.

The effect of acylation of goat alpha-lactalbumin on lactose synthetase activity and the ability of alpha-lactalbumin to inhibit the transfer of galactose to N-acetylglucosamine is biphasic. Approx. 15% of the lactose synthase activity of goat alpha-lactalbumin and 10% of its inhibitory power is lost in the initial phase, with corresponding losses of 65 and 30% in the second phase. Deacylation of reacted tyrosyl groups with hydroxylamine restored inhibitory power completely in the initial phase and partially in the second phase. Removal of acyl groups in the initial phase decreased lactose synthase activity, but had no effect in the second phase. The differential effect of acylation of alpha-lactalbumin on lactose synthase and inhibitory properties appears to be the result of differential changes in the affinity of the UDP-Gal-galactosyl-transferase-alpha-lactalbumin ternary complex for monosaccharides.

Impairment of the catalytic activity of Escherichia coli ribonucleic acid polymerase by a unique reaction of 4-chloro-7-nitrobenzofurazan.

Escherichia coli RNA polymerase loses 55-65% of its catalytic activity on reaction with Nbf-Cl (4-choro-7-nitrobenzofurazan). This partial inactivation was shown to be the result of specific impairment of RNA-chain elongation, since initiation of RNA chains was not altered after treatment with Nbf-Cl. The site of reaction was shown to be a unique thiol on the beta-subunit. This thiol is not accessible to reaction with 5,5'-dithiobis-(2-nitrobenzoic acid). No protection of the enzyme against reaction with Nbf-Cl could be obtained with the inhibitor rifamycin nor with calf thymus DNA, GTP or 1,10-phenanthroline, indicating that the unique thiol is probably not within the active site. The specific impairment of RNA-chain elongation thus appears to be the result of a local conformational change which leaves chain initiation unimpaired. Changes observed in the tryptophan fluorescence spectrum of the enzyme or reaction with Nbf-Cl are consistent with formation of a Meisenheimer complex of the reagent with a nucleophilic group on the enzyme near the reactive thiol. It is proposed that formation of such a complex and a subsequent conformational change renders this thiol unusually susceptible to reaction with Nbf-Cl.

Anomalous reaction of 4-chloro-7-nitrobenzofurazan with thiol compounds.

The kinetics of reaction of 4-chloro-7-nitrobenzofurazan with thiol groups at pH values above 5 cannot be accounted for solely on the basis of formation of a single product, the 4-thio derivative. Spectroscopic observations indicate that, in addition to the 4-thio derivative, at least two other products are formed. One of these, referred to as P1, is most likely a reversible complex of thiol compound and 4-chloro-7-nitrobenzofurazan of the Meisenheimer type. The other product, P2, which forms primarily when thiol compound is in a large excess, does not appear to result from direct reaction of thiol group and 4-chloro-7-nitrobenzofurazan, but may be a reaction of product P1 and thiol compound. The coloured product, P2, will react further with proteins, such as bovine serum albumin and Escherichia coli RNA polymerase. This reaction irreversibly destroys the catalytic activity of RNA polymerase. The implications of these observations for utilization of 4-chloro-7-nitrobenzofurazan as a protein-modifying agent are discussed.