Rosenstiel Basic Medical Sciences Research Center, MS029, Brandeis University, Waltham, MA 02454-9110 and 1 Department of Molecular and Cell Biology, Division of Biochemistry and Molecular Biology, University of California, Berkeley, CA 94720, USA
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Abstract |
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Keywords: aspartate aminotransferase/conserved cysteine/crystal structure/maleate/pyridoxal phosphate
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Introduction |
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The roles of the five cysteines found in the primary structure of L-Asp-AT from E.coli were examined by mutagenesis (Gloss et al., 1992). Only Cys191 is conserved in all of the closely related transaminase sequences (>25% sequence identity, including L-Asp-AT and E.coli tyrosine aminotransferase). A residue that is conserved in eubacteria, yeast, birds and mammals is likely to play an important role in catalysis, folding or stability, and mutation of this residue is likely to affect structure and/or function adversely. However, the two-base mutation, C191A, yielded an active and stable enzyme, with properties very similar to those of the wild-type enzyme. The single base mutation, C191S, was also active and stable, but exhibited some striking kinetic differences from the wild-type enzyme. The hypothesis was advanced that Cys191 was conserved, not because it is essential, but because there is no neutral mutational corridor to the phenotypically similar enzyme, Ala191 (Gloss et al., 1992
).
In a subsequent study, all possible single base mutations at position 191 (Cys to Phe, Tyr, Trp, Arg and Gly, as well as Ser) were made and characterized to test further this evolutionary hypothesis (Gloss et al., 1996). All of the L-Asp-AT variants are active and stable, but have significant decreases in some kinetic or stability parameters, relative to wild type or C191A, supporting the evolutionary hypothesis (Table I
). The most striking difference observed in the set of single base mutations was that four mutations, C191S, C191F, C191Y and C191W, affected the pKa of the internal aldimine between the PLP cofactor and Lys258. For the wild-type and C191A enzyme, this pKa is approximately 7, whereas these four mutations resulted in an alkaline shift of 0.60.8 pH units for this pKa. This pKa value is reflected in the pH-dependence of kcat/Km for aspartate of the enzymes (Kiick and Cook, 1983
; Gloss and Kirsch, 1995a
). At physiological pH, the kcat/Km Asp values of wild type and C191S are very similar; the values for C191F, C191Y and C191W are 2.44-fold lower than that of wild type. This difference in the kcat/Km Asp values is smaller at the optimal pH values of each enzyme, pH 8.28.6 (Gloss et al., 1996
). It was also noted that replacing a buried cysteine with a large aromatic side chain (C191F, Y, W) had only a marginal effect on protein stability (0.30.6 kcal/mol decrease).
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Materials and methods |
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Mutant E.coli L-Asp AT was prepared as described previously (Gloss et al., 1996) and appeared as a single band on a Coomassie-stained SDS polyacrylamide gel. The best crystals were obtained at room temperature by the hanging-drop vapor diffusion method in 24 well Lindbro dishes with siliconized cover slips. Hanging drops contained 3 µl protein solution and 3 µl reservoir solution, and were allowed to equilibrate against 1 ml reservoir solution. The final crystallization conditions contained 613 mg/ml L-Asp-AT (7, 13, 6 and 9 mg/ml for the C191S, C191F, C191Y and C191W mutants, respectively), 20 mM potassium phosphate buffer, pH 7.5, 10 µM PLP and 5 mM EDTA in the protein solution. The reservoir buffers contained 4054% ammonium sulfate and 20 mM potassium phosphate buffer, pH 7.5. Some crystals (for the C191S + maleate and the C191F, C191Y and C191W structures) were grown in the presence of the competitive inhibitor maleate (5 mM). All the crystals were bright yellow due to the presence of the PLP cofactor. Crystals used for data collection were rectangular rods approximately 0.3 mm in diameter and between 0.5 and 1.0 mm in length.
Data collection
Single crystals were mounted in thin-walled quartz capillary tubes. X-Ray diffraction data were collected at 4°C with an oscillation range of 1° per frame with 30 min exposures, using a 0.3 mm collimator on a R-Axis IIC imaging plate system. CuK radiation
was provided by a Rigaku RU200-HB rotating anode generator operated at 50 kV and 145 mA. For the C191F, C191Y and C191W structures and the C191S structure with maleate, still photos were taken and used in indexing to determine unit cell parameters, and the data frames were integrated, scaled and merged with standard R-Axis software (Wonacott, 1980
; Higashi, 1990
). For the C191S structure without maleate, 1° oscillation photos were taken and used in indexing to determine unit cell parameters, and the data frames were integrated, scaled and merged with DENZO (Otwinowski and Minor, 1997
). The symmetry of each diffraction pattern in the photos used for indexing was consistent with the orthorhombic space group C2221.
Data reduction and model refinement
C191S mutant with maleate
A native data set was obtained at a crystal to detector distance of 130 mm. The Rmerge was 5.9% on intensities for all reflections (Table II). The final merged data set contained 17 708 unique reflections with I > 0, corresponding to 86% completeness in a resolution range of 10.02.45 Å. The protein coordinates from an aspartate aminotransferase structure, from which the water molecule coordinates were removed, were used as the initial model (PDB entry 1ASA; Smith et al., 1989). The model was refined with XPLOR (Brünger et al., 1987
; Brünger, 1990
, 1996
) against all reflections between 10.0 and 2.45 Å resolution. The final model has an R-factor of 20.7% and contains 26 water molecules.
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C191F mutant with maleate
A native data set was obtained at a crystal to detector distance of 100 mm. The Rmerge was 3.8% on intensities for all reflections (Table II). The final merged data set contained 40 823 unique reflections with I > 0, corresponding to 90% completeness in a resolution range of 10.01.9 Å. The protein coordinates from a partially refined C191S with maleate structure were used as the initial model. The model was refined with XPLOR against all reflections between 10.0 and 1.90 Å resolution. The final model has an R-factor of 18.8% and contains 144 water molecules.
C191Y mutant with maleate
A native data set was obtained at a crystal to detector distance of 80 mm. The Rmerge was 6.0% on intensities for all reflections (Table II). The final merged data set contained 34 520 unique reflections with I > 0, corresponding to 80% completeness in a resolution range of 10.02.2 Å. The protein coordinates from a partially refined C191S with maleate structure were used as the initial model. The model was refined with XPLOR against all reflections between 10.0 and 2.20 Å resolution. The final model has an R-factor of 18.5% and contains 80 water molecules.
C191W mutant with maleate
A native data set was obtained at a crystal to detector distance of 80 mm. The Rmerge was 4.2% on intensities for all reflections (Table II). The final merged data set contained 38 214 unique reflections with I > 0, corresponding to 80% completeness in a resolution range of 10.01.90 Å. The protein coordinates from a partially refined C191S with maleate structure were used as the initial model. The model was refined with XPLOR against all reflections between 10.0 and 1.90 Å resolution. The final model has an R-factor of 18.9% and contains 117 water molecules.
C191S mutant without maleate
A native data set was obtained at a crystal to detector distance of 100 mm. The Rmerge was 7.2% on intensities for all reflections (Table II). The final merged data set contained 19 539 unique reflections with I > 0, corresponding to 92% completeness in a resolution range of 10.02.40 Å. The protein coordinates from a wild type E.coli L-Asp-AT structure were used as an initial model (PDB entry 1AAW; Almo et al., 1994). The model was refined with XPLOR against all reflections between 10.0 and 2.40 Å resolution. Water molecules were placed manually using a difference Fourier map with the coefficients |Fo Fc| and a 2.0
cut-off. The final model has an R-factor of 21.9% and contains 120 water molecules.
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Results and discussion |
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Each of the mutant structures has the same overall fold as the wild-type protein (Jäger et al., 1994) with each subunit consisting of a large and a small domain and an N-terminal segment that extends over the other subunit in the dimer. Each of the five structures contains a PLP cofactor covalently bound in the active site of each monomer through a Schiff base linkage to the
amino group of lysine 258. Four of the structures also contain a maleic acid inhibitor in the active site and are found to be in the `closed' form of the protein (C191W, C191F, C191Y and C191S with maleate). The fifth structure does not contain a maleic acid inhibitor and is found to be in the `open' form of the protein (C191S without maleate).
Differences between the wild-type protein and the mutant proteins
The general shape of the active site and the location of the active site residues relative to the maleic acid inhibitor and the PLP cofactor are largely unchanged compared with what is observed for wild-type L-Asp-AT. When the PLP cofactors in the structures are superposed, the nearby active site residues superpose closely (Figure 2). One exception is the distance between the side chain of Tyr225 and the O3' of the PLP cofactor in the open forms of the C191S mutant and wild-type structures. In the wild-type enzyme, the side chain hydroxyl group of Tyr225 is in a position to make a good hydrogen bond (2.5 Å) with the O3' of the PLP cofactor. In the C191S mutant structures, this distance is increased by 0.4 Å (Table III
).
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Since one of the C191S structures was solved in the absence of bound inhibitor, we can use it to help explain the observed shift in the pKa of the enzyme compared with the wild-type enzyme. The change in the hydrogen bonding pattern between residues 191 and 225 results in a shift of the side chain of active site residue Tyr225 so that its hydroxyl group moves away from the PLP oxygen (from 2.5 to 2.9 Å; Table III). The change in the normally close interaction of residue 225 with the PLP cofactor appears to explain the effect of the substitutions at Cys191 on the pKa of the internal aldimine. The longer distance between the Tyr225 hydroxyl group and the PLP oxygen means that the Tyr225 side chain makes a weaker hydrogen bond with the oxygen. The weaker hydrogen bond then results in the 0.69 pH unit alkaline shift of the pKa of the internal aldimine that has been reported previously (Table I
) (Gloss et al., 1996
). This is consistent with the results seen previously with a Tyr225 to Phe225 mutant enzyme (Goldberg et al., 1991
) where the hydrogen bond between the Tyr225 side chain and the PLP oxygen is absent and there is a 1.3 pH unit alkaline shift in pKa.
Accommodation of the aromatic side chains
While the serine substitution results in a small rotation of the side chain of residue 191, the corresponding phenylalanine, tyrosine and tryptophan side chains in the C191F, C191Y and C191W mutant proteins are rotated by almost 180° around the CCß bond relative to C191 in the wild-type structure. This rotation places the large aromatic side chains into a pre-existing pocket composed of the side chains of residues Asp199, Arg230, Asn357, Trp205 and backbone atoms from residues 236 to 239. This pocket is large enough so that none of the residues lining the pocket are within 3 Å of the mutant side chain. The presence of the pocket allows the large aromatic side chain to be accommodated in the three-dimensional structure without a significant change in the positions of nearby backbone or side chain atoms. One side of the pocket is accessible to solvent, and each of the aromatic side chains is partially solvent-accessible. The solvent- accessible surface area calculated with a probe that is 2 Å in diameter is 8.8 Å2 for the phenylalanine side chain, 13.9 Å2 for the tyrosine side chain and 11.2 Å2 for the tryptophan side chain.
Conclusions
This set of mutant enzymes provides an example of how catalysis can be altered by a single amino acid substitution at a position located outside of the active site and without changes in the overall structure or large decreases in the stability of the enzyme. It emphasizes the importance of amino acid residues near the active site that do not interact directly with the cofactor or substrate (or in this case, inhibitor). These residues may be important in helping to position precisely the functional groups in the active site, because, as is seen here, a change in the length of even one hydrogen bond between an active site residue and a cofactor can affect catalysis.
Coordinates
Atomic coordinates have been deposited in the Protein Data Bank with accession codes 5eaa, 1b4x, 1qir, 1qis and 1qit.
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Acknowledgments |
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Notes |
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References |
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Received May 7, 1999; revised November 15, 1999; accepted November 15, 1999.