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.     

Mutation of a single amino acid converts germ cell alkaline phosphatase to placental alkaline phosphatase

Human placental and germ cell alkaline phosphatases (PLAP and GCAP, respectively), are characterized by their differential sensitivities to inhibition by L-leucine, EDTA, and heat. Yet, they differ by only 7 amino acids at positions 15, 67, 68, 84, 241, 254, and 429 within their respective 484 residues. To determine the structural basis and the amino acid(s) involved in these physicochemical differences, we constructed three GCAP mutants by site-directed mutagenesis and six GCAP/PLAP chimeras and then expressed these alkaline phosphatase mutants in COS-1 cells. We report that the differential reactivity of PLAP and GCAP depends critically on a single amino acid at position 429. GCAP with Gly-429 is strongly inhibited by L-leucine, EDTA, and heat, whereas PLAP with Glu-429 is resistant. By substituting Gly-429 of GCAP with a series of amino acids, we demonstrate that the relative sensitivities of these mutants to L-leucine, EDTA, and heat inhibition are, in general, parallel. Mutants in the order of resistance to these treatments are: Glu (most resistant), Asp/Ile/Leu, Gln/Val/Lys, Ser/His, and Arg/Thr/Met/Cys/Phe/Trp/Tyr/Pro/Asn/Ala/Gly (least resistant). However, the Ser-429 and His-429 mutants were more resistant to EDTA and heat inhibition than the wild-type GCAP, but were equally sensitive to L-leucine inhibition. Structural analysis of mammalian alkaline phosphatase modeled on the refined crystal structure of Escherichia coli alkaline phosphatase indicates that the negative charge of Glu-429 of PLAP, which simultaneously stabilizes the protein as a whole and the metal binding specifically, probably acts through interactions with the metal ligand His-320 (His-331 in E. coli alkaline phosphatase). Replacement of codon 429 with Gly in GCAP leads to destabilization and loosening of the metal binding. The data suggest that the natural binding site for L-leucine may be near position 429, with the amino and carboxyl groups of L-leucine interacting with bound phosphate and His-432 (His-412 in E. coli alkaline phosphatase), respectively.     

A single amino acid substitution in B subunit of Escherichia coli enterotoxin affects its oligomer formation

We isolated a mutant strain of enterotoxigenic Escherichia coli by nitrosoguanidine mutagenesis, which produces an immunologically altered B subunit of heat-labile enterotoxin. This mutant B subunit was detected as a monomer on sodium dodecyl sulfate-polyacrylamide gel electrophoresis even without prior heating, suggesting a problem in oligomer formation. Furthermore, this mutant B subunit could not form holotoxin with the native A subunit, and the affinity to GM1-ganglioside receptor was 10-fold lower than that of the native B subunit. The amino acid sequence analysis of this mutant B subunit revealed only one amino acid substitution compared with the native B subunit, at the 64th position from the N terminus (valine instead of alanine). These data suggest that the alanine at position 64 from the N terminus is important for the native B subunit to form an oligomer structure and express its functions.     

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.     

Molecular asymmetry in alkaline phosphatase of Escherichia coli

Thermal inactivation of alkaline phosphatase of Escherichia coli has been studied at different temperatures (45 to 70 degrees C) and pHs (7.5, 9.0, and 10.0) for the commercial, buffer-dialyzed (pH 9.0) and EDTA-dialyzed (pH 9.0) enzymes. In each case, the inactivation exhibits biphasic kinetics consistent with the rate equation, (formula; see text) where A0 and A are activities at time zero and t, and k1 and k2 are first-order rate constants for the fast and slow phase, respectively. Values of k1 and k2 change independently with temperature, pH, and pretreatment (dialysis) of the enzyme. Time course of inactivation of the enzyme with excess EDTA and effect of Zn2+ ion concentration on the activity of EDTA-dialyzed enzyme have been investigated. The data suggest that the dimeric enzyme protein has two types of catalytic sites which have equal catalytic efficiency (or specific activity) but differ in several other properties. Structural implications of these results have been discussed.     

Studies on alkaline phosphatase. Transientstate and steady-state kinetics of Escherichia coli alkaline phosphatase

1. Reduction of a 19s immunoglobulin M with 3mm-mercaptoethanol or 0.05-0.5mm-dithiothreitol followed by alkylation gave sedimentation patterns indicating products compatible with structures consisting of one, two, three, four and five 7s sub-units. This supports the concept of a five-sub-unit structure for immunoglobulin M. 2. Reduction with 0.125mm-dithiothreitol or 20mm-cysteine produced 7s sub-units that could not be dissociated into chains in m-propionic acid. 3. By labelling (with iodo[2-(14)C]acetic acid) the thiol groups liberated during reduction with 0.125mm-dithiothreitol, it was possible to identify the tryptic peptides involved in the disulphide bridges that link the 7s sub-units together (inter-sub-unit bridges). 4. By further reducing and labelling (with iodo[2-(14)C]acetic acid) the 7s sub-units produced by 0.125mm-dithiothreitol, it was possible to identify tryptic peptides derived from intra-sub-unit bridges. 5. Sub-units produced by reduction with 20mm-cysteine proved to be unsuitable for distinguishing between inter-sub-unit bridges and intra-sub-unit bridges. 6. The possible arrangement of the interchain disulphide bridges was deduced.     

A single amino acid substitution in the active site of Escherichia coli aspartate transcarbamoylase prevents the allosteric transition

Modeling of the tetrahedral intermediate within the active site of Escherichia coli aspartate transcarbamoylase revealed a specific interaction with the side-chain of Gln137, an interaction not previously observed in the structure of the X-ray enzyme in the presence of N-phosphonacetyl-L-aspartate (PALA). Previous site-specific mutagenesis experiments showed that when Gln137 was replaced by alanine, the resulting mutant enzyme (Q137A) exhibited approximately 50-fold less activity than the wild-type enzyme, exhibited no homotropic cooperativity, and the binding of both carbamoyl phosphate and aspartate were extremely compromised. To elucidate the structural alterations in the mutant enzyme that might lead to such pronounced changes in kinetic and binding properties, the Q137A enzyme was studied by time-resolved, small-angle X-ray scattering and its structure was determined in the presence of PALA to 2.7 angstroms resolution. Time-resolved, small-angle X-ray scattering established that the natural substrates, carbamoyl phosphate and L-aspartate, do not induce in the Q137A enzyme the same conformational changes as observed for the wild-type enzyme, although the scattering pattern of the Q137A and wild-type enzymes in the presence of PALA were identical. The overall structure of the Q137A enzyme is similar to that of the R-state structure of wild-type enzyme with PALA bound. However, there are differences in the manner by which the Q137A enzyme coordinates PALA, especially in the side-chain positions of Arg105 and His134. The replacement of Gln137 by Ala also has a dramatic effect on the electrostatics of the active site. These data taken together suggest that the side-chain of Gln137 in the wild-type enzyme is required for the binding of carbamoyl phosphate in the proper orientation so as to induce conformational changes required for the creation of the high-affinity aspartate-binding site. The inability of carbamoyl phosphate to create the high-affinity binding site in the Q137A enzyme results in an enzyme locked in the low-activity low-affinity T state. These results emphasize the absolute requirement of the binding of carbamoyl phosphate for the creation of the high-affinity aspartate-binding site and for inducing the homotropic cooperativity in aspartate transcarbamoylase.     

Cell envelope protection of alkaline phosphatase against acid denaturation in Escherichia coli.

The effects of a highly acidic environment on the cell-associated alkaline phosphatase activities of a smooth and a rough strain of Escherichia coli O8 have been examined. The observation that cell-associated enzyme is denatured to a lesser degree than purified enzyme suggests that the association of the enzyme with the cell envelope affords it some degree of protection from potentially disruptive agents in the environment. The degree of protection afforded the enzyme from pH denaturation appears to be dependent upon the presence of a complete lipopolysaccharide in the outer membrane of these strains. An abbreviation of the chemical structure of this cell envelope component produces a change in the outer membrane, resulting in increased susceptibility of the cells to a battery of antibiotics and to lysozyme and in a small, but significant, change in the sensitivity of the cell envelope-associated alkaline phosphatase to the denaturing effect of an acidic environment.     

A single amino acid substitution in the A subunit of Escherichia coli enterotoxin results in a loss of its toxic activity

A plasmid encoding a mutant gene of heat-labile enterotoxin (LT), produced by enterotoxigenic Escherichia coli, was induced by treatment of plasmid EWD 299 with hydroxylamine. A mutant strain of E. coli HB 101 carrying the mutant plasmid pTUH 6A produced a low toxic LT analogue (mutant LT), which was cross-reactive with anti-LT antibody. The mutant LT activity was less than 0.15 and 0.006% of the normal LT in the rabbit ileal loop test and in the rabbit skin permeability test, respectively. The amino acid composition of the mutant LT-B subunit was the same as that of the normal B subunit. Though the A2 fragment of the mutant LT was identical to normal LT by DNA analysis, the A1 fragment of the mutant LT differed from the normal A1 fragment in one amino acid at position 112; namely it had lysine instead of glutamic acid from the N terminus. These data suggest that glutamic acid at position 112 from the N terminus of the A1 fragment is important for the A subunit to express its biological activity.     

Involvement of tryptophan 209 in the allosteric interactions of Escherichia coli aspartate transcarbamylase using single amino acid substitution mutants

Five mutant versions of aspartate transcarbamylase have been isolated, all with single amino acid substitutions in the catalytic chain of the enzyme. A previously isolated pyrB nonsense mutant was suppressed with supB, supC, supD and supG to create enzymes with glutamine, tyrosine, serine or lysine, respectively, inserted at the position of the nonsense codon. Each of these enzymes was purified to homogeneity and kinetically characterized. The approximate location of the substitution was determined by using tryptic fingerprints of the wild-type enzyme and the enzyme obtained with a tyrosine residue inserted at the position of the nonsense codon. By first cloning the pyrBI operon, from the original pyrB nonsense strain, followed by sequencing of the appropriate portion of the gene, the exact location of the mutation was determined to be at position 209 of the catalytic chain. Site-directed mutagenesis was used to generate versions of aspartate transcarbamylase with tyrosine and glutamic acid at this position. The Tyr209 enzyme is identical with that obtained by suppression of the original nonsense mutation with supC. The two enzymes produced by site-directed mutagenesis were purified using a newly created overproducing strain. Kinetic analysis revealed that each mutant has an altered affinity for aspartate, as judged by variations in the substrate concentration at one-half maximal activity. In addition, the mutants exhibit altered Hill coefficients and maximal activities. In the wild-type enzyme, position 209 is a tryptophan residue that is involved in the stabilization of a bend in the molecule near the subunit interface region. The alteration in homotropic cooperativity seems to be due to changes induced in this bend in the molecule, which stabilizes alternate conformational states of the enzyme.