Jürgen Kirchberger

Methods for the separation of lactate dehydrogenases and clinical significance of the enzyme

Lactate dehydrogenase (LDH), an ubiquitous enzyme among vertebrates, invertebrates, plants and microbes was discovered in the early period of enzymology. The enzyme has been dissolved in several distinguishable molecular forms. In mammals, three types of subunits encoded by the genes Ldh-A, Ldh-B and Ldh-C give rise to a selected number of tetrameric isoenzymes. LDH-A4, LDH-B4 and the mixed hybrid forms of the A- and B-subunits are present in many tissues but with certain distribution patterns. LDH-C4 is confined in mammals to testes and sperm. Numerous techniques have been employed to purify, characterize and separate the different forms of the enzyme. This report deals with the main protocols and procedures of purification of LDH and its isoenzymes including chromatographic and electrophoretic methods, partitioning in aqueous two-phase systems and precipitation approaches. In particular, affinity separation techniques based on natural and pseudo-biospecific ligands are described in detail. In addition, basic physico-chemical and kinetic properties of the enzyme from different sources are summarized in a second part, the clinical significance of the determination of LDH in diverse body fluids in respect to the total activity and the isoenzyme distribution in different organs is discussed.

The constitutive activity of the adhesion GPCR GPR114/ADGRG5 is mediated by its tethered agonist.

Adhesion GPCRs (aGPCRs) form the second largest, yet most enigmatic class of the GPCR super‐family. Although the physiologic importance of aGPCRs was demonstrated in several studies, the majority of these receptors is still orphan with respect to their agonists and signal transduction. Recent studies reported that aGPCRs are activated through a tethered peptide agonist, coined the Stachel sequence. The Stachel sequence is the most C‐terminal part of the highly conserved GPCR autoproteolysis‐inducing domain. Here, we used cell culture‐based assays to investigate 2 natural splice variants within the Stachel sequence of the orphan Gs coupling aGPCR GPR114/ADGRG5. There is 1 variant constitutively active in cAMP assays (~ 25‐fold over empty vector) and sensitive to mechano‐activation. The other variant has low basal activity in cAMP assays (6‐fold over empty vector) and is insensitive to mechano‐activation. In‐depth mutagenesis studies of these functional differences revealed that the N‐terminal half of the Stachel sequence confers the agonistic activity, whereas the C‐terminal part orientates the agonistic core sequence to the transmembrane domain. Sequence comparison and functional testing suggest that the proposed mechanism of Stachel‐mediated activation is relevant not only to GPR114 but to aGPCRs in general.—Wilde, C., Fischer, L., Lede, V., Kirchberger, J., Rothemund, S., Schöneberg, T., Liebscher, I. The constitutive activity of the adhesion GPCR GPR114/ADGRG5 is mediated by its tethered agonist. FASEB J. 30, 666‐673 (2016). www.fasebj.org

Preparation of Homogeneous Alkaline Phosphatase 211011 Galf Intestine by Dye-Ligand Chromatography

The paper deals with a simple and effective procedure for the isolation of calf intestinal alkaline phosphatase (EC 3.1.3.1) with a yield of 35 per cent by employing immobilized Procion Red HE-3B and Cibacron Blue F3G-A, respectively, as dye-ligands. The resulting enzyme is homogeneous and has a specific activity of about 2500 units per mg of protein. Because dye liganded gels are of low costs and can be used several times without loss of binding properties, the presented method is in particular suited for large scale application.

Structure and allosteric regulation of eukaryotic 6-phosphofructokinases.

Abstract Although the crystal structures of prokaryotic 6-phosphofructokinase, a key enzyme of glycolysis, have been available for almost 25 years now, structural information about the more complex and highly regulated eukaryotic enzymes is still lacking until now. This review provides an overview of the current knowledge of eukaryotic 6-phosphofructokinase based on recent crystal structures, kinetic analyses and site-directed mutagenesis data with special focus on the molecular architecture and the structural basis of allosteric regulation.

Assembly of phosphofructokinase-1 from Saccharomyces cerevisiae in extracts of single-deletion mutants

Phosphofructokinase‐1 from Saccharomyces cerevisiae is an octameric enzyme comprising two non‐identical subunits, α and β, which are encoded by the unlinked genes PFK1 and PFK2. In this paper, assembly and reactivation of the enzyme have been studied in cell‐free extracts of single‐deletion mutants. In contrast to the previously described lack of phosphofructokinase‐1 activity in cell‐free extracts of these mutants, we could measure a temporary enzyme activity immediately after lysis of protoplasts. This result supports the assumption that each of the subunits forms an enzyme structure which is active in vivo but not stable after cell disruption.

Molecular architecture and structural basis of allosteric regulation of eukaryotic phosphofructokinases

Eukaryotic ATP‐dependent 6‐phospho‐fructokinases (Pfks) differ from their bacterial counterparts in a much more complex structural organization and allosteric regulation. Pichia pastoris Pfk (PpPfk) is, with ~1 MDa, the most complex and probably largest eukaryotic Pfk. We have determined the crystal structure of full‐length PpPfk to 3.05 Å resolution in the T state. PpPfk forms a (αβγ)4 dodec‐amer of D2 symmetry with dimensions of 161 × 157 × 233 Å mainly via interactions of the α chains. The N‐terminal domains of the a and β chains have folds that are distantly related to glyoxalase I, but the active sites are no longer functional. Interestingly, these domains located at the 2 distal ends of this protein along the long 2‐fold axis form a (aβ)2 dimer as does the core Pfk domains;however, the domains are swapped across the tetramerization interface. In PpPfk, the unique γ subunit participates in oligomerization of the αβ chains. This modulator protein was acquired from an ancient S‐adenosylmethionine‐dependent methyl‐transferase. The identification of novel ATP binding sites, which do not correspond to the bacterial catalytic or effector binding sites, point to marked structural and functional differences between bacterial and eukaryotic Pfks.—Strater, N., Marek, S., Kuettner, E. B., Kloos, M., Keim, A., Brüser, A., Kirch‐berger, J., Schoneberg, T. Molecular architecture and structural basis of allosteric regulation of eukary‐otic phosphofructokinases. FASEB J. 25, 89–98 (2011). www.fasebj.org

Interaction of 6-phosphofructokinase with cytosolic proteins of Saccharomyces cerevisiae

Hetero‐octameric 6‐phosphofructokinase (Pfk‐1) from Saccharomyces cerevisiae is composed of two types of subunits, α and β, which are encoded by the unlinked genes PFK1 and PFK2. Pfk single deletion mutants expressing only one type of subunit exhibit Pfk‐1 activity in vivo which, however, is completely lost immediately after cell disruption. In order to elucidate the preconditions of the in vivo activity of the mutant enzymes composed of either α‐ or β‐subunits, we have investigated their potential interaction with selected heat shock and cytoskeletal proteins, employing co‐immunoprecipitation and immunofluorescence microscopy. Western blot analysis identified the mitochondrial chaperonin Hsp60, as well as the cytoskeleton proteins α‐tubulin and actin, in complexes with Pfk‐1 that were co‐precipitated from a cell‐free extract of a pfk2 single deletion mutant expressing only the α‐subunit. The interaction of the corresponding mutant enzyme and Hsp60 was found to depend on the ATP concentration of the extract. Immunofluorescence microscopy displayed a conspicuously filamentous arrangement of the Pfk‐1 mutant protein, exclusively in the pfk2 single deletion mutant. The analysis of structure and activity of Pfk‐1 expressed in S. cerevisiae mutant strains defective in various heat shock proteins (TRiC/CCT, Hsp70, Hsp 104) and in the respective wild‐type background did not reveal significant differences. Copyright

Functional Linkage of Adenine Nucleotide Binding Sites in Mammalian Muscle 6-Phosphofructokinase *

Background: Crystal structures of 6-phosphofructokinases revealed nucleotide binding sites with unknown functional relevance. Results: Function of two allosteric nucleotide binding sites was determined by mutagenesis and kinetic studies and revealed reciprocal linkage of both. Conclusion: Activity of mammalian Pfk is regulated by structurally linked new allosteric sites. Significance: Reciprocal linkage between allosteric binding sites evolved convergent in prokaryotes and eukaryotes. 6-Phosphofructokinases (Pfk) are homo- and heterooligomeric, allosteric enzymes that catalyze one of the rate-limiting steps of the glycolysis: the phosphorylation of fructose 6-phosphate at position 1. Pfk activity is modulated by a number of regulators including adenine nucleotides. Recent crystal structures from eukaryotic Pfk revealed several adenine nucleotide binding sites. Herein, we determined the functional relevance of two adenine nucleotide binding sites through site-directed mutagenesis and enzyme kinetic studies. Subsequent characterization of Pfk mutants allowed the identification of the activating (AMP, ADP) and inhibitory (ATP, ADP) allosteric binding sites. Mutation of one binding site reciprocally influenced the allosteric regulation through nucleotides interacting with the other binding site. Such reciprocal linkage between the activating and inhibitory binding sites is in agreement with current models of allosteric enzyme regulation. Because the allosteric nucleotide binding sites in eukaryotic Pfk did not evolve from prokaryotic ancestors, reciprocal linkage of functionally opposed allosteric binding sites must have developed independently in prokaryotic and eukaryotic Pfk (convergent evolution).

A Novel Form of 6-Phosphofructokinase IDENTIFICATION AND FUNCTIONAL RELEVANCE OF A THIRD TYPE OF SUBUNIT IN PICHIA PASTORIS

Classically, 6-phosphofructokinases are homo- and hetero-oligomeric enzymes consisting of α subunits and α/β subunits, respectively. Herein, we describe a new form of 6-phosphofructokinase (Pfk) present in several Pichia species, which is composed of three different types of subunit, α, β, and γ. The sequence of the γ subunit shows no similarity to classic Pfk subunits or to other known protein sequences. In-depth structural and functional studies revealed that the γ subunit is a constitutive component of Pfk from Pichia pastoris (PpPfk). Analyses of the purified PpPfk suggest a heterododecameric assembly from the three different subunits. Accordingly, it is the largest and most complex Pfk identified yet. Although, the γ subunit is not required for enzymatic activity, the γ subunit-deficient mutant displays a decreased growth on nutrient limitation and reduced cell flocculation when compared with the P. pastoris wild-type strain. Subsequent characterization of purified Pfks from wild-type and γ subunit-deficient strains revealed that the allosteric regulation of the PpPfk by ATP, fructose 2,6-bisphosphate, and AMP is fine-tuned by the γ subunit. Therefore, we suggest that the γ subunit contributes to adaptation of P. pastoris to energy resources.

6‐phosphofructokinase from Pichia pastoris: purification, kinetic and molecular characterization of the enzyme

6‐Phosphofructokinase from Pichia pastoris was purified for the first time to homogeneity applying seven steps, including pseudo‐affinity dye‐ligand chromatography on Procion Blue H‐5R‐Sepharose. The specific activity of the purified enzyme was about 80 U/mg. It behaves as a typically allosteric 6‐phosphofructokinase exhibiting activation by AMP and fructose 2,6‐bis(phosphate), inhibition by ATP and cooperativity to fructose 6‐phosphate. However, in comparison with the enzymes from Saccharomyces cerevisiae and Kluyveromyces lactis, the activation ratio of 6‐phosphofructokinase from Pichia pastoris by AMP is several times higher, the ATP inhibition is stronger and the apparent affinity to fructose 6‐phosphate is significantly lower. Aqueous two‐phase affinity partitioning with Cibacron Blue F3G‐A did not reflect remarkable structural differences of the nucleotide binding sites of the Pfks from Pichia pastoris and Saccharomyces cerevisiae. The structural organisation of the active enzyme seems to be different in comparison with hetero‐octameric 6‐phosphofructokinases from other yeast species. The enzyme was found to be a hetero‐oligomer with an molecular mass of 975 kDa (sedimentation equilibrium measurements) consisting of two distinct types of subunits in an equimolar ratio with molecular masses of 113 kDa and 98 kDa (SDS–PAGE), respectively, and a third non‐covalently complexed protein component (34 kDa, SDS–PAGE). The latter seems to be necessary for the catalytic activity of the enzyme. Sequencing of the N‐terminus (VTKDSIXRDLEXENXGXXFF) and of peptide fragments by applying MALDI–TOF PSD, m/z 1517.3 (DAMNVVNH) and m/z 2177.2 [AQNCNVC(L/I)SVHEAHTM] gave no relevant information about the identity of this protein. Copyright