Biomedical subjects

P T Beernink

Publications and source records attributed to P T Beernink.

9 recordsLinked to original sources

PubMed2001

Structural basis for a change in substrate specificity: crystal structure of S113E isocitrate dehydrogenase in a complex with isopropylmalate, Mg2+, and NADP.

Isocitrate dehydrogenase (IDH) catalyzes the oxidative decarboxylation of isocitrate and has negligible activity toward other (R)-malate-type substrates. The S113E mutant of IDH significantly improves its ability to utilize isopropylmalate as a substrate and switches the substrate specificity (k(cat)/K(M)) from isocitrate to isopropylmalate. To understand the structural basis for this switch in substrate specificity, we have determined the crystal structure of IDH S113E in a complex with isopropylmalate, NADP, and Mg(2+) to 2.0 A resolution. On the basis of a comparison with previously determined structures, we identify distinct changes caused by the amino acid substitution and by the binding of substrates. The S113E complex exhibits alterations in global and active site conformations compared with other IDH structures that include loop and helix conformational changes near the active site. In addition, the angle of the hinge that relates the two domains was altered in this structure, which suggests that the S113E substitution and the binding of substrates act together to promote catalysis of isopropylmalate. Ligand binding results in reorientation of the active site helix that contains residues 113 through 116. E113 exhibits new interactions, including van der Waals contacts with the isopropyl group of isopropylmalate and a hydrogen bond with N115, which in turn forms a hydrogen bond with NADP. In addition, the loop and helix regions that bind NADP are altered, as is the loop that connects the NADP binding region to the active site helix, changing the relationship between substrates and enzyme. In combination, these interactions appear to provide the basis for the switch in substrate specificity.

Amino Acid Substitution↗

PubMed2001

Two divalent metal ions in the active site of a new crystal form of human apurinic/apyrimidinic endonuclease, Ape1: implications for the catalytic mechanism.

The major human abasic endonuclease, Ape1, is an essential DNA repair enzyme that initiates the removal of apurinic/apyrimidinic sites from DNA, excises 3' replication-blocking moieties, and modulates the DNA binding activity of several transcriptional regulators. We have determined the X-ray structure of the full-length human Ape1 enzyme in two new crystal forms, one at neutral and one at acidic pH. The new structures are generally similar to the previously determined structure of a truncated Ape1 protein, but differ in the conformation of several loop regions and in spans of residues with weak electron density. While only one active-site metal ion is present in the structure determined at low pH, the structure determined from a crystal grown at the pH optimum of Ape1 nuclease activity, pH 7.5, has two metal ions bound 5 A apart in the active site. Enzyme kinetic data indicate that at least two metal-binding sites are functionally important, since Ca(2+) exhibits complex stimulatory and inhibitory effects on the Mg(2+)-dependent catalysis of Ape1, even though Ca(2+) itself does not serve as a cofactor. In conjunction, the structural and kinetic data suggest that Ape1 catalyzes hydrolysis of the DNA backbone through a two metal ion-mediated mechanism.

Binding Sites↗

PubMed2001

Random circular permutation leading to chain disruption within and near alpha helices in the catalytic chains of aspartate transcarbamoylase: effects on assembly, stability, and function.

A collection of circularly permuted catalytic chains of aspartate transcarbamoylase (ATCase) has been generated by random circular permutation of the pyrB gene. From the library of ATCases containing permuted polypeptide chains, we have chosen for further investigation nine ATCase variants whose catalytic chains have termini located within or close to an alpha helix. All of the variants fold and assemble into dodecameric holoenzymes with similar sedimentation coefficients and slightly reduced thermal stabilities. Those variants disrupted within three different helical regions in the wild-type structure show no detectable enzyme activity and no apparent binding of the bisubstrate analog N:-phosphonacetyl-L-aspartate. In contrast, two variants whose termini are just within or adjacent to other alpha helices are catalytically active and allosteric. As expected, helical disruptions are more destabilizing than loop disruptions. Nonetheless, some catalytic chains lacking continuity within helical regions can assemble into stable holoenzymes comprising six catalytic and six regulatory chains. For seven of the variants, continuity within the helices in the catalytic chains is important for enzyme activity but not necessary for proper folding, assembly, and stability of the holoenzyme.

Amino Acid Sequence↗

PubMed2000

Binding of bisubstrate analog promotes large structural changes in the unregulated catalytic trimer of aspartate transcarbamoylase: implications for allosteric regulation.

A central problem in understanding enzyme regulation is to define the conformational states that account for allosteric changes in catalytic activity. For Escherichia coli aspartate transcarbamoylase (ATCase; EC) the active, relaxed (R state) holoenzyme is generally assumed to be represented by the crystal structure of the complex of the holoenzyme with the bisubstrate analog N-phosphonacetyl-L-aspartate (PALA). It is unclear, however, which conformational differences between the unliganded, inactive, taut (T state) holoenzyme and the PALA complex are attributable to localized effects of inhibitor binding as contrasted to the allosteric transition. To define the conformational changes in the isolated, nonallosteric C trimer resulting from the binding of PALA, we determined the 1.95-A resolution crystal structure of the C trimer-PALA complex. In contrast to the free C trimer, the PALA-bound trimer exhibits approximate threefold symmetry. Conformational changes in the C trimer upon PALA binding include ordering of two active site loops and closure of the hinge relating the N- and C-terminal domains. The C trimer-PALA structure closely resembles the liganded C subunits in the PALA-bound holoenzyme. This similarity suggests that the pronounced hinge closure and other changes promoted by PALA binding to the holoenzyme are stabilized by ligand binding. Consequently, the conformational changes attributable to the allosteric transition of the holoenzyme remain to be defined.

Allosteric Regulation↗

PubMed1999

Assessment of the allosteric mechanism of aspartate transcarbamoylase based on the crystalline structure of the unregulated catalytic subunit.

The lack of knowledge of the three-dimensional structure of the trimeric, catalytic (C) subunit of aspartate transcarbamoylase (ATCase) has impeded understanding of the allosteric regulation of this enzyme and left unresolved the mechanism by which the active, unregulated C trimers are inactivated on incorporation into the unliganded (taut or T state) holoenzyme. Surprisingly, the isolated C trimer, based on the 1.9-A crystal structure reported here, resembles more closely the trimers in the T state enzyme than in the holoenzyme:bisubstrate-analog complex, which has been considered as the active, relaxed (R) state enzyme. Unlike the C trimer in either the T state or bisubstrate-analog-bound holoenzyme, the isolated C trimer lacks 3-fold symmetry, and the active sites are partially disordered. The flexibility of the C trimer, contrasted to the highly constrained T state ATCase, suggests that regulation of the holoenzyme involves modulating the potential for conformational changes essential for catalysis. Large differences in structure between the active C trimer and the holoenzyme:bisubstrate-analog complex call into question the view that this complex represents the activated R state of ATCase.

Allosteric Regulation↗

PubMed1996

Disruption of the aldolase A tetramer into catalytically active monomers.

The fructose-1,6-bisphosphate aldolase (EC 4.1.2.13) homotetramer has been destabilized by site-directed mutagenesis at the two different subunit interfaces. A double mutant aldolase, Q125D/E224A, sediments as two distinct species, characteristic of a slow equilibrium, with velocities expected for the monomer and tetramer. The aldolase monomer is shown to be catalytically active following isolation from sucrose density gradients. The isolated aldolase monomer had 72% of the specific activity of the wild-type enzyme and a slightly lower Michaelis constant, clearly indicating that the quaternary structure is not required for catalysis. Cross-linking of the isolated monomer confirmed that it does not rapidly reequilibrate with the tetramer following isolation. There was a substantial difference between the tetramer and monomer in their inactivation by urea. The stability toward both urea and thermal inactivation of these oligomeric variants suggests a role for the quaternary structure in maintaining the stability of aldolase, which may be an important role of quaternary structure in many proteins.

Amino Acid Sequence↗

PubMed1994

Subunit interface mutants of rabbit muscle aldolase form active dimers.

We report the construction of subunit interface mutants of rabbit muscle aldolase A with altered quaternary structure. A mutation has been described that causes nonspherocytic hemolytic anemia and produces a thermolabile aldolase (Kishi H et al., 1987, Proc Natl Acad Sci USA 84:8623-8627). The disease arises from substitution of Gly for Asp-128, a residue at the subunit interface of human aldolase A. To elucidate the role of this residue in the highly homologous rabbit aldolase A, site-directed mutagenesis is used to replace Asp-128 with Gly, Ala, Asn, Gln, or Val. Rabbit aldolase D128G purified from Escherichia coli is found to be similar to human D128G by kinetic analysis, CD, and thermal inactivation assays. All of the mutant rabbit aldolases are similar to the wild-type rabbit enzyme in secondary structure and kinetic properties. In contrast, whereas the wild-type enzyme is a tetramer, chemical crosslinking and gel filtration indicate that a new dimeric species exists for the mutants. In sedimentation velocity experiments, the mutant enzymes as mixtures of dimer and tetramer at 4 degrees C. Sedimentation at 20 degrees C shows that the mutant enzymes are > 99.5% dimeric and, in the presence of substrate, that the dimeric species is active. Differential scanning calorimetry demonstrates that Tm values of the mutant enzymes are decreased by 12 degrees C compared to wild-type enzyme. The results indicate that Asp-128 is important for interface stability and suggest that 1 role of the quaternary structure of aldolase is to provide thermostability.

Anemia, Hemolytic↗

PubMed1992

Construction of a high-copy "ATG vector" for expression in Escherichia coli.

We report the construction of an inducible, high-copy plasmid for the expression of foreign proteins in Escherichia coli. This plasmid, pPB1, combines the trc promoter, beta-galactosidase translation start site, and polylinker of pKK233-2 with the origin of replication region of pUC19. Replacement of the origin of replication of pKK233-2 results in a threefold increase in plasmid copy number of pPB1 compared with pKK233-2. Subclones of the cDNA for rabbit muscle fructose-1,6-bisphosphate aldolase (E.C. 4.1.2.13) in the two expression plasmids exhibit a comparable difference in copy number. An increase in protein expression measured by SDS-PAGE and aldolase specific activities reflects the increased copy number. Specific activities of aldolases in bacterial extracts differ approximately sixfold between the two expression plasmids in E. coli JM83. Aldolase A can compose up to 40% of the total protein in E. coli JM83 when expressed in pPB1, from which more than 100 mg of purified enzyme can be obtained per liter culture.

Base Sequence↗

PubMedDate not supplied

High-throughput, fluorescence-based screening for soluble protein expression.

Protein expression screening methods are essential for proteomic scale characterization of gene and cDNA expression libraries. Screening methods are also important for the identification of highly expressed protein targets, for example, in quantities suitable for high-throughput screening and protein structural studies. To address these needs, we describe the implementation of several rapid, fluorescence-based protein expression screening strategies using Escherichia coli or E. coli-based in vitro transcription/translation (IVT) systems. In vitro expression screening is fast, convenient and, as we show, correlates well with in vivo expression. For screening, expressed proteins are labeled either as fusions with green fluorescent protein (GFP) or through translational incorporation of a fluorescent amino acid derivative, BODIPY-FL-Lysine. Fluorescence-based detection of GFP fusions or BODIPY-labeled proteins is considerably faster than other common expression screening methods, such as immunological detection of gels or dot blots. Furthermore, in vitro and in vivo screening used together yield a larger set of expressed proteins than either method alone. Specifically labeled proteins in cellular lysates are detected in one of three formats: a microplate using a fluorescence plate reader, a dot-blot using a fluorescence scanner or a microarray using a laser scanner. We have established a correlation among the various detection formats, which validates the use of protein microarrays for expression screening. Production of expressed proteins detected through screening can be scaled up either using IVT reactions or with in vivo expression systems in the absence of a fluorophore for subsequent characterization of protein function or interactions.

Boron Compounds↗