Influence of cofactor pyridoxal 5'-phosphate on reversible high-pressure denaturation of isolated .beta.2 dimer of tryptophan synthase bienzyme complex from Escherichia coli

Seifert, Thomas; Bartholmes, Peter; Jaenicke, Rainer

doi:10.1021/bi00323a016

Cited by 29 publications

(16 citation statements)

References 37 publications

(43 reference statements)

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“…PLP-induced stabilization has been observed previously for many, but not all, PLP-binding enzymes. For example, rat liver mitochondrial aspartate aminotransferase, O-acetylserine sulfhydrylase, glutamate decarboxylase, sheep liver serine hydroxymethyltransferase, and tryptophan synthase were found to be stabilized by PLP (33)(34)(35)(36)(37)(38), but no stabilization has been observed for serine hydroxymethyltransferase (39) or dopa decarboxylase (40).…”

Section: Discussionmentioning

confidence: 99%

In Vivo and in Vitro Examination of Stability of Primary Hyperoxaluria-associated Human Alanine:Glyoxylate Aminotransferase

Hopper

Pittman

Fitzgerald

et al. 2008

Journal of Biological Chemistry

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Primary hyperoxaluria type I is a severe kidney stone disease caused by mutations in the protein alanine:glyoxylate aminotransferase (AGT). Many patients have mutations in AGT that are not deleterious alone but act synergistically with a common minor allele polymorphic variant to impair protein folding, dimerization, or localization. Although studies suggest that the minor allele variant itself is destabilized, no direct stability studies have been carried out. In this report, we analyze AGT function and stability using three approaches. First, we describe a yeast complementation growth assay for AGT, in which we show that human AGT can substitute for function of yeast Agx1 and that mutations associated with disease in humans show reduced growth in yeast. The reduced growth of minor allele mutants reflects reduced protein levels, indicating that these proteins are less stable than wild-type AGT in yeast. We further examine stability of AGT alleles in vitro using two direct methods, a mass spectrometry-based technique (stability of unpurified proteins from rates of H/D exchange) and differential scanning fluorimetry. We also examine the effect of known ligands pyridoxal 5-phosphate and aminooxyacetic acid on stability. Our work establishes that the minor allele is destabilized and that pyridoxal 5-phosphate and aminooxyacetic acid binding significantly stabilizes both alleles. To our knowledge, this is the first work that directly measures relative stabilities of AGT variants and ligand complexes. Because previous studies suggest that stabilizing compounds (i.e. pharmacological chaperones) may be effective for treatment of primary hyperoxaluria, we propose that the methods described here can be used in high throughput screens for compounds that stabilize AGT mutants.Deficiencies in the enzyme alanine:glyoxylate aminotransferase (AGT) 3 cause primary hyperoxaluria type I (PH1), a severe autosomal recessive kidney stone disease (1, 2). In humans, AGT is responsible for conversion of glyoxylate to glycine in the liver. Without functional AGT, glyoxylate builds up and is converted to calcium oxalate, which is deposited in the kidneys and can lead to kidney stones and renal failure. In many patients, deficiency of AGT results from one or two amino acid changes that decrease the stability of this enzyme. As a result, AGT may be degraded, become improperly localized, or form nonfunctional aggregates (1, 2).Over 50 different mutations of AGT and two polymorphic variants have been identified (1, 3). The two allelic forms consist of a "wild-type" major allele, AGTma, and a minor allele, AGTmi. The minor allele is present in ϳ20% of European and North American populations and contains P11L and I340M substitutions in the amino acid sequence and a 74-bp duplication in intron 1 (4, 5). The P11L and I340M substitutions, particularly P11L, have several biochemical effects, including decreasing catalytic activity and slowing the dimerization rate, but by themselves are not disease-causing (4, 5). The minor allele of AGT is delete...

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Section: Discussionmentioning

confidence: 99%

In Vivo and in Vitro Examination of Stability of Primary Hyperoxaluria-associated Human Alanine:Glyoxylate Aminotransferase

Hopper

Pittman

Fitzgerald

et al. 2008

Journal of Biological Chemistry

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“…The ratio of the initial velocity to the steady-state velocity was determined for each protease concentration and is reported adjacent to individual progress curves as a percent with initial protease concentration. The change in activity with time for each protease concentration was determined at various time intervals by drawing tangents to the curves and fitting the following equation for reversible dimer dissociation (23) Proc. Natl.…”

Section: Methodsmentioning

confidence: 99%

Stability and activity of human immunodeficiency virus protease: comparison of the natural dimer with a homologous, single-chain tethered dimer.

Cheng

Yin

Foundling

et al. 1990

Proc. Natl. Acad. Sci. U.S.A.

117

101

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“…Weber and colleagues (20)(21)(22)(23)(24) and others (25) have studied the dissociation of various oligomeric proteins by high pressure and subsequent reassembly at atmospheric pressure. The association constants reported for dimeric proteins are similar in magnitude to the value we report for CAP.…”

Section: Methodsmentioning

confidence: 99%

Modulation of the stability of a gene-regulatory protein dimer by DNA and cAMP.

Brown

Crothers

1989

Proc. Natl. Acad. Sci. U.S.A.

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We describe an experimental approach to the measurement of protein subunit exchange in which biotinylated subunits mediate attachment of35S-labeled subunits to a streptavidin column as a result of the exchange process. Application of the method to Escherichia coli catabolite activator protein (CAP) revealed that in the absence of cAMP, the dimerization equilibrium constant is 3 x 1010 M-', with a dimer lifetime of 300 min. Exchange of CAP subunits is accelerated at least 1000-fold by the presence of nonspecific DNA, under low ionic strength conditions. Catalysis of exchange also occurs at physiological ionic conditions. In contrast, physiological concentrations of cAMP stabilize CAP with respect to subunit exchange in either the presence or the absence of DNA. We discuss the functional implications of monomerization of gene-regulatory proteins resulting from kinetic and thermodynamic lability of their dimers.During the past several years there has been rapid growth of structural information about gene-regulatory proteins, particularly prokaryotic helix-turn-helix proteins (1, 2). X-ray crystal structures have been reported for at least two ligandregulated proteins, catabolite activator protein (CAP) and trp repressor (1,3,4), and for several other regulatory proteins (5, 6). These molecules have in common a homodimeric composition, generally thought to be an efficient motif by which to gain greater binding affinity and specificity for their respective pseudosymmetric DNA binding sequences (7). Subunit interchange of dimeric regulatory proteins has been postulated in order to explain phenomena ranging from transcriptional regulation by heterodimers (8, 9) to catalysis by DNA of the dissociation of a protein-DNA complex by stepwise removal of the monomers (10).CAP is a homodimeric protein at physiological ionic strength. The ability of CAP to stimulate DNA-dependent transcription by Escherichia coli RNA polymerase is cAMPdependent. In the absence of polymerase, cAMP stimulates sequence-specific binding of CAP to the DNA region upstream of the promoter; without cAMP only weaker, nonspecific DNA binding is observed. The dimer interaction in CAP is through hydrophobic surfaces of identical subunits, and each of the two cAMP binding sites are composed of elements of both subunits (3). Gel electrophoresis studies have indicated that the dissociation of specific CAPpromoter complexes displays a second-order dependence on competing DNA concentration. One of the mechanisms proposed involves sequential removal of monomers from the specific complex by nonspecific competitor DNA (10).In this article we describe a direct assay to test this hypothesis, employing an approach in which biotinylated CAP subunits act to retain 35S-labeled subunits to a streptavidin column as a result of subunit exchange. The assay was employed to assess the effect ofnatural ligands such as cAMP and DNA. We find that DNA alone destabilizes the CAP dimer, whereas cAMP has a stabilizing effect. These results lead us to propose that cAMP regul...

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Influence of cofactor pyridoxal 5'-phosphate on reversible high-pressure denaturation of isolated .beta.2 dimer of tryptophan synthase bienzyme complex from Escherichia coli

Cited by 29 publications

References 37 publications

In Vivo and in Vitro Examination of Stability of Primary Hyperoxaluria-associated Human Alanine:Glyoxylate Aminotransferase

In Vivo and in Vitro Examination of Stability of Primary Hyperoxaluria-associated Human Alanine:Glyoxylate Aminotransferase

Stability and activity of human immunodeficiency virus protease: comparison of the natural dimer with a homologous, single-chain tethered dimer.

Modulation of the stability of a gene-regulatory protein dimer by DNA and cAMP.

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