PubMed Health⌕ Search

Biomedical subjects

H Gehring

Publications and source records attributed to H Gehring.

At least 55 records · Page 3Linked to original sources
PubMed1996

[Plasma volume determination with ICG dye in changes of intravascular volume. Dye dilution method before and after autologous blood donation and after retransfusion in the human].

OBJECTIVE: The determination of the current intravascular plasma volume with the dye dilution method is of major interest in therapeutic intervention and scientific research. The use of the rapidly eliminated dye indocyanine green (ICG) allows to perform plasma volume measurements serially and to observe a time course. The following questions should be investigated in the present study: 1. Do the plasma volume (PV) values measured by the ICG method in this study agree with data of other authors with special respect to basic values for further clinical investigations, and 2. Do the measured differences coincide with the withdrawn and retransfused plasma volume? DESIGN: Prospective study (on each 20 healthy female and male volunteers). SETTING: Research laboratory of a university department of anesthesiology. PARTICIPANTS: Each 20 healthy female and male volunteers. INTERVENTIONS: Measurement of PV with ICG both before and after withdrawal of 10% of blood volume and after retransfusion. RESULTS: The plasma volume per body surface area (PV/BSA) was in the group of female volunteers 1639 +/- 198 and in male volunteers 1687 +/- 224 ml/m2 (mean +/- SD). The withdrawn amount of plasma volume (EPV) was 188 +/- 23 in the female group and 149 +/- 26 ml/m2 in the male group. Determined by the ICG method the difference for the withdrawal was in female volunteers 198 +/- 174 and in male volunteers 171 +/- 158 ml/m2 and for the retransfusion in the female group 190 +/- 169 and in the male group 142 +/- 154 ml/m2. CONCLUSIONS: The mean differences in plasma volume measured by the ICG method both before and after withdrawal and after retransfusion of autologous blood were in good agreement with the withdrawn amount of plasma volume. The large standard deviation has to be considered in clinical assessment of an individual measurement.

Adult↗
PubMed1995

Changing the reaction specificity of a pyridoxal-5'-phosphate-dependent enzyme.

The electron distribution in the coenzyme-substrate adduct of aspartate aminotransferase was changed by replacing active-site Arg386 with alanine and introducing a new arginine residue nearby. [Y225R, R386A]Aspartate aminotransferase decarboxylates L-aspartate to L-alanine (kcat = 0.04 s-1), while its transaminase activity towards dicarboxylic amino acids is decreased by three orders of magnitude (kcat = 0.19 s-1). Molecular-dynamics simulations based on the crystal structure of the mutant enzyme suggest that a new hydrogen bond to the imine N atom of the pyridoxal-5'-phosphate- aspartate adduct and an altered electrostatic potential around its beta-carboxylate group underlie the 650,000-fold increase in the ratio of beta-decarboxylase/transaminase activity.

Aspartate Aminotransferases↗
PubMed1995

Structural basis for the catalytic activity of aspartate aminotransferase K258H lacking the pyridoxal 5'-phosphate-binding lysine residue.

Chicken mitochondrial and Escherichia coli aspartate aminotransferases K258H, in which the active site lysine residue has been exchanged for a histidine residue, retain partial catalytic competence [Ziak et al. (1993) Eur. J. Biochem. 211, 475-484]. Mutant PLP and PMP holoenzymes and the complexes of the latter (E. coli enzyme) with sulfate and 2-oxoglutarate, as well as complexes of the mitochondrial apoenzyme with N-(5'-phosphopyridoxyl)-L-aspartate or N-(5'-phosphopyridoxyl)-L-glutamate, were crystallized and analyzed by means of X-ray crystallography in order to examine how the side chain of histidine 258 can substitute as a general acid/base catalyst of the aldimine-ketimine tautomerization in enzymic transamination. The structures have been solved and refined at resolutions between 2.1 and 2.8 A. Both the closed and the open conformations, identical to those of the wild-type enzyme, were observed, indicating that the mutant enzymes of both species exhibit the same conformational flexibility as the wild-type enzymes, although in AspAT K258H the equilibrium is somewhat shifted toward the open conformation. The replacement of the active site K258 by a histidine residue resulted only in local structural adaptations necessary to accommodate the imidazole ring. The catalytic competence of the mutant enzyme, which in the forward half-reaction is 0.1% of that of the wild-type enzyme, suggests that the imidazole group is involved in the aldimine-ketimine tautomerization. However, the imidazole ring of H258 is too far away from C alpha and C4' of the coenzyme-substrate adduct for direct proton transfer, suggesting that the 1,3-prototropic shift is mediated by a water molecule. Although there is enough space for a water molecule in this area, it has not been detected. Dynamic fluctuations of the protein matrix might transiently open a channel, giving a water molecule fleeting access to the active site.

Animals↗
PubMed1994

Modulation of the activity of mitochondrial aspartate aminotransferase H352C by the redox state of the engineered interdomain disulfide bond.

Molecular modeling suggested that the large and small domain of mitochondrial aspartate aminotransferase might be linked by an engineered disulfide bond that could be expected to interfere with ligand-induced and syncatalytic changes in conformation and thus to assist in the elucidation of their significance for the catalytic mechanism. His-352, which is situated in the small domain close to Cys-166 of the large domain, was replaced with a cysteine residue by oligonucleotide-directed mutagenesis. Aspartate aminotransferase H352C, that had not been exposed to reducing conditions, in part contained a disulfide bond between Cys-166 and Cys-352. Exposure to a reducing agent cleaved the crosslink completely and produced an enzyme derivative with 8% of the activity of the wild type enzyme. Cu2+-mediated autoxidation resulted in complete formation of the disulfide bond and a decrease in enzymic activity to 2%. Independently of the redox state of the disulfide bond, the H352C substitution seems to shift the equilibrium from the open toward the closed conformation of the enzyme. This change in conformation was accompanied by an increase in the binding affinity for both the amino and oxo acid substrate by one order of magnitude. Apparently, 1-2 kcal/mol of the binding energy of the substrates are no longer diverted to shift the conformational equilibrium toward the closed conformation. The kcat/Km values were unchanged or even increased in the reduced form of the mutant enzyme and only slightly decreased in its oxidized form. Both the disulfide-independent decrease in enzymic activity, as observed in reduced aspartate aminotransferase H352C and also in two other mutant enzymes (C166H/H352C and H352Q), and the redox-dependent modulation of activity indicate that unhindered domain movements are essential for full catalytic competence of aspartate aminotransferase.

Animals↗
PubMed1994

Shift in pH-rate profile and enhanced discrimination between dicarboxylic and aromatic substrates in mitochondrial aspartate aminotransferase Y70H.

Tyr70 of chicken mitochondrial aspartate aminotransferase was replaced with a histidine residue by oligonucleotide-directed mutagenesis. Aspartate aminotransferase Y70H retained at pH 7.5 13% of the activity toward dicarboxylic amino acids, whereas the activity toward aromatic amino acids was only 0.6% of that of the wild-type enzyme, corresponding to a 22-fold increase in the ratio of the activities toward these two types of substrates. In comparison to that of the wild-type enzyme, the low-pH limb of the pH-activity profile of the mutant enzyme was shifted to higher pH values, very likely reflecting the titration curve of the newly introduced histidine residue with a pKa' of 6.3. Apparently, a positively charged residue at position 70 abolishes enzymic activity. The spectrophotometrically determined pKa' value of the internal aldimine formed between pyridoxal 5'-phosphate and Lys258 in the mutant enzyme was 6.0, similar to that in the wild-type enzyme. The rate constant of the dissociation of pyridoxamine 5'-phosphate from the mutant enzyme was increased only 3 times over that of the wild-type enzyme, in contrast to the 80-fold increase in Escherichia coli aspartate aminotransferase Y70F [Toney, M. D., & Kirsch, J. F. (1987) J. Biol. Chem. 262, 12403-12405], suggesting that His70 can replace Tyr70 in forming a hydrogen bond to the coenzyme.

Amino Acid Sequence↗
PubMed1994

Kinetics of molecular chaperone action.

Molecular chaperones of the Hsp70 type transiently sequester unfolded segments of proteins and promote their correct folding. Target peptides were labeled with an environmentally sensitive fluorophore so that their binding to the molecular chaperone DnaK of Escherichia coli could be followed in real time. The two-step process was characterized by relaxation times of 27 seconds and 200 seconds with 2 microM DnaK and 0.1 microM ligand at 25 degrees C. In the presence of adenosine triphosphate, the formation of the complex was greatly accelerated and appeared to be a single-exponential process with a relaxation time of 0.4 second. The binding-release cycle of DnaK thus occurs in the time range of polypeptide chain elongation and folding and is too fast to be stoichiometrically coupled to the adenosine triphosphatase activity of the chaperone (turnover number, 0.13 per minute at 30 degrees C).

2-Naphthylamine↗
PubMed1994

Probing conformational states of spin-labeled aspartate aminotransferase by ESR.

Mitochondrial aspartate aminotransferase was selectively labeled with various maleimide-linked nitroxide spin labels at the conformationally sensitive Cys166. The mobility of the spin group was found to increase with increasing length of the spacer between the nitroxide and maleimide moiety. The label with the ethylcarbamoyl group, a spacer of intermediate length, responded sensitively to conformational changes of aspartate aminotransferase. The modification with this label decreased the enzymic activity to 30% of its initial value and increased the affinity for various substrates and inhibitors 5-10-fold. Identical ESR spectra were obtained for the pyridoxal and pyridoxamine form of the enzyme. These spectra are complex, consisting of an isotropic and at least two anisotropic components. The spectral complexity is attributed to different modes of interaction of the spin label with its local protein environment giving rise to different motional states. The same changes in the ESR spectra have been observed upon formation of the adsorption complex of the pyridoxal form with a competitive inhibitor and on formation of covalent intermediates of the transamination reaction. Essentially, the isotropic component is converted to a new anisotropic one as the local environment changes due to a conformational adaptation of aspartate aminotransferase. The ESR data are consistent with an equilibrium between two conformational states of the enzyme but inconsistent with individual protein conformations of the various intermediates of the transamination reaction. The two conformational states may be assigned to the open and closed conformations as defined by X-ray crystallography. In the adsorption complex of the pyridoxal enzyme, and in the covalent intermediates, the two-state equilibrium appears to be shifted towards the closed conformation in which the spin label is more rigidly bound, as also suggested by molecular dynamic simulations of the label modelled into aspartate aminotransferase. In contrast the formation of adsorption complexes between the pyridoxamine form and aspartate or maleate was not accompanied by the same shift of the conformational equilibrium.

Aspartate Aminotransferases↗
PubMed1993

The mature form of imported mitochondrial proteins undergoes conformational changes upon binding to isolated mitochondria.

Mature mitochondrial proteins (aspartate aminotransferase, malate dehydrogenase, hydroxyacyl coenzyme A dehydrogenase, creatine kinase) and cytosolic proteins (aldolase, glyceraldehyde-3-phosphate dehydrogenase) with a basic pI were found to bind to isolated mitochondria, electrostatic interactions being mainly responsible for their binding. Mitochondrial aspartate aminotransferase bound with a Kd' of 30 nM in 0.6 M sorbitol, 20 mM Hepes/KOH, pH 7.4, at 25 degrees C. Cytosolic aspartate aminotransferase and glutamate dehydrogenase (a protein located in the mitochondrial matrix) both with an acidic pI, did not bind to mitochondria. Treatment of mitochondria with proteinases did not affect the subsequent binding of imported mitochondrial proteins. Their association with both intact and proteinase-treated mitochondria resulted in a marked increase in their susceptibility toward proteinase K. In contrast, the basic cytosolic proteins tested bound only to intact mitochondria and thereby did not become more susceptible toward proteolytic attack. Treatment of mitochondria with adriamycin, a drug binding to acidic phospholipids, prevented the subsequent association of mitochondrial aspartate aminotransferase with mitochondria and the ensuing conformational labilization. Apparently, the mature moiety of imported mitochondrial proteins is partially unfolded upon interaction with the lipid component of the mitochondrial envelope. Both the binding of the mitochondrial proteins and their conformational labilization is independent of ATP and the electrochemical potential across the inner membrane.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1993

Mutant aspartate aminotransferase (K258H) without pyridoxal-5'-phosphate-binding lysine residue. Structural and catalytic properties.

If the pyridoxal-phosphate-binding lysine residue 258 of aspartate aminotransferase is exchanged for a histidine residue, the enzyme retains partial catalytic competence [Ziak, M., Jaussi, R., Gehring, H. and Christen, P. (1990) Eur. J. Biochem. 187, 329-333]. The three-dimensional structures of the mutant enzymes of both chicken mitochondria and Escherichia coli were determined at high resolution. The folding patterns of the polypeptide chains proved to be identical to those of the wild-type enzymes, small conformational differences being restricted to parts of the active site. If aspartate or glutamate was added to the pyridoxal form of the mutant enzyme [lambda max 392 nm and 330 nm (weak); negative CD at 420 nm, positive CD at 370 nm and 330 nm], the external aldimine (lambda max = 430 nm; negative CD at 360 nm and 430 nm) transiently accumulated. Upon addition of 2-oxoglutarate to the pyridoxamine form (lambda max 330 nm, positive CD), a putative ketamine intermediate could be detected; however, with oxalacetate, an equilibrium between external aldimine and the pyridoxal form, which was strongly in favour of the former, was established within seconds. The transamination cycle with glutamate and oxalacetate proceeds only three orders of magnitude more slowly than the overall reaction of the wild-type enzyme. The specific activity of the mutant enzyme is 0.1 U/mg at 25 degrees C and constant from pH 6.0 to 8.5. Reconstitution of the mutant apoenzyme with [4'-3H]pyridoxamine 5'-phosphate resulted in rapid release of 3H with a first-order rate constant kappa' = 5 x 10(-4) s-1 similar to that of the wild-type enzyme. Apparently, in aspartate aminotransferase, histidine can to some extent substitute for the active-site lysine residue. The imidazole ring of H258, however, seems too distant from C alpha and C4' to act efficiently as proton donor/acceptor in the aldimine-ketamine tautomerization, suggesting that the prototropic shift might be mediated by an intervening water molecule. Transmination of the internal to the external aldimine apparently can be replaced by de novo formation of the latter, and by its hydrolysis in the reverse direction.

Animals↗
PubMed1993

CDGF (chicken embryo fibroblast-derived growth factor) is mitogenically related to TGF-beta and modulates PDGF, bFGF, and IGF-I action on sparse NIH/3T3 cells.

Chicken embryo fibroblast (CEF)-derived growth factor (CDGF), which was recently isolated from serum-free conditioned medium (SFCM) of confluent primary cultures of CEF (A. Geistlich and H. Gehring, Eur. J. Biochem. 207, 147-153, 1992), exhibited a strong mitogenic activity on sparse cultures of NIH/3T3 cells. The activity of CDGF was different from that of SFCM; i.e., the onset of DNA synthesis was delayed for about 7 h. CDGF induced maximally 25% of the activity of serum or SFCM if the activity was measured with a 2-h[3H]thymidine pulse starting 15 h after stimulation of the cells, indicating loss of a protein which modulated the mitogenic activity of CDGF. However, [3H]-thymidine incorporation of cells stimulated with approximately 50 pM CDGF reached serum values after prolongation of the thymidine pulse to 24 h. PDGF, at a concentration of approximately 300 pM, and bFGF (approximately 10 pM) exhibited strong activities in the 2-h pulse, whereas TGF-beta behaved like CDGF. IGF-I induced [3H]thymidine incorporation only weakly and only in the 2-h pulse. EGF did not induce any [3H]thymidine incorporation at all. CDGF together with small concentrations of bFGF (3.5 pM), higher concentrations of PDGF (300 pM), or IGF-I (1 nM) increased synergistically thymidine incorporation in the 2-h pulse, exceeding in the case of PDGF and bFGF the values obtained with 10% serum. Such a synergism could not be demonstrated with alpha-fetoprotein or fetuin, two serum proteins which have been reported to cooperate with growth factors. Regarding induction of cellular growth, only PDGF proved similar to serum, whereas cells stimulated with CDGF or TGF-beta showed a decreased rate of multiplication during the first day after stimulation. After this lag, however, CDGF- and TGF-beta-stimulated cells grew also with a rate similar to that obtained with serum, indicating the induction of an autocrine mitogen by CDGF or TGF-beta. FGF, IGF-I, and PDGF all enhanced CDGF-induced cell growth during the first day, whereas an additive stimulation over at least 2 days was observed with PDGF. CDGF behaved similar to TGF-beta in the synergism with IGF-I.

3T3 Cells↗
PubMed1992

Isolation and characterization of a novel type of growth factor derived from serum-free conditioned medium of chicken embryo fibroblasts.

A strong mitogenic activity for fibroblastic cells has been found in serum-free medium of growth-arrested primary cultures of chicken embryo fibroblasts (CEF). This serum-free conditioned medium promoted growth of NIH/3T3 cells and primary as well as secondary cultures of CEF. The mitogenic activity was as potent as 5% serum. Half-maximum stimulation was obtained with 20% of the initial concentration of the conditioned medium. The activity eluted at high M(r) (1-2 x 10(5)) from a gel-filtration column under nondenaturing conditions and was trypsin insensitive and thiol insensitive. Treatment with acid or urea converted the mitogen to a low-molecular-mass form, which showed a delayed induction of DNA synthesis. Purification of this factor (10000-fold) to apparent homogeneity was achieved by preparative isoelectric focusing, gel filtration, reverse-phase HPLC and nonreducing SDS/PAGE. The factor, termed CEF-derived growth factor (CDGF) was a 32-kDa, disulfide-linked heterodimer of a 15-kDa and a 17-kDa subunit as judged by SDS/PAGE, with a pI of approximately 7 in 8 M urea. It exhibited partial stability towards heat treatment and was trypsin sensitive. CDGF was active only in its dimeric form and half-maximum stimulation of NIH/3T3 cells was obtained at approximately 10 pM. The mitogenic activity was not suppressible by an antibody neutralizing the activity of transforming growth factor beta 1, 2 and 3 (TGF-beta). The physico-chemical properties suggest that CDGF is not identical with one of the common growth factors like fibroblast growth factor, platelet-derived growth factor, epidermal growth factor, insulin-like growth factor, or TGF-beta but rather represents a novel type of growth factor.

3T3 Cells↗
PubMed1991

Spectroscopic characterization of true enzyme-substrate intermediates of aspartate aminotransferase trapped at subzero temperatures.

Absorption and circular dichroism spectra of stable enzyme-substrate intermediates of aspartate aminotransferase were recorded at subzero temperatures (down to -65 degrees C) in the cryosolvent water/methanol. The intermediates were formed either between the pyridoxal form of the enzyme and its amino acid substrates, or between the pyridoxamine form and its oxo acid substrates. Kd values determined by spectroscopic titration were very close to the Km values reported for the different substrates. The adsorption complex of the pyridoxal form was probably obtained on addition of cysteine sulfinate. This complex is characterized by an increased absorption at 430 nm together with a positive Cotton effect, as also observed in the case of the complex with the competitive inhibitor maleate indicating protonation of the internal aldimine. Addition of the substrates aspartate or glutamate to the pyridoxal form seemed to result in the direct accumulation of the external aldimine which showed a slight decrease in both the absorbance and the Cotton effect at 360 nm. Additionally, a bathochromic shift of 5 nm was observed in the case of glutamate. At 430 nm, only a minor increase in absorbance, but not in circular dichroism, was observed with aspartate, and no changes were found with glutamate and the substrate analog 2-methylaspartate, indicating a deprotonated external aldimine. Presumably, the ketimine intermediate was obtained on addition of the oxo acids 2-oxoglutarate or oxalacetate to the pyridoxamine form. The intermediate showed a slight bathochromic shift (2 nm) of the absorption band and decreased circular dichroism. On formation of the ketimine, a tyrosine residue, probably active-site Tyr225, becomes partly ionized. The finding that the external aldimine can probably be accumulated in the conversion of the pyridoxal to the pyridoxamine form with the natural substrates would confirm the proton abstraction at C alpha to be the rate-limiting step in the tautomerization, although with cysteine sulfinate, the formation of the external aldimine might contribute to the rate limitation. Accumulation of the ketimine in the reverse direction would indicate that the proton abstraction at C4' is rate-limiting in this half-reaction. The results demonstrate the feasibility of further structural investigations of true enzyme-substrate intermediates.

Animals↗
PubMed1991

Thymidine secretion by cultured chicken embryo fibroblasts and NIH/3T3 cells: quantification and time course.

Cultured chicken embryo fibroblasts (CEF) as well as mouse NIH/3T3 fibroblasts secrete substantial amounts of thymidine into the medium. The rate of secretion is maximal when the cells become confluent. In the medium of confluent primary cultures of CEF up to 10 microM thymidine was measured. Lower concentrations (1 to 2 microM) were found in media of NIH/3T3 cultures. In media conditioned by NIH/3T3 cells, the degradation product thymine was also found, which was not detectable in the sera and the media of CEF. Moderate inhibitory effects on the growth of CEF and NIH/3T3 cells (at least 10% with a relatively large range of variation) indicated that thymidine (2 to 5 microM) might contribute to cellular homeostasis. The secretion of high amounts of endogenous thymidine into cell culture media may falsify the assessment of DNA synthesis with labeled thymidine; thus a change of the medium is recommended when performing a thymidine pulse.

Animals↗
PubMed1990

Aspartate aminotransferase with the pyridoxal-5'-phosphate-binding lysine residue replaced by histidine retains partial catalytic competence.

The active site residue lysine 258 of chicken mitochondrial aspartate aminotransferase was replaced with a histidine residue by means of site-directed mutagenesis. The mutant protein was expressed in Escherichia coli and purified to homogeneity. Addition of 2-oxoglutarate to its pyridoxamine form changed the coenzyme absorption spectrum (lambda max = 330 nm) to that of the pyridoxal form (lambda max = 330/392 nm). The rate of this half-reaction of transamination (kcat = 4.0 x 10(-4)s-1) is five orders of magnitude slower than that of the wild-type enzyme. However, the reverse half-reaction, initiated by addition of aspartate or glutamate to the pyridoxal form of the mutant enzyme, is only three orders of magnitude slower than that of the wild-type enzyme, kmax of the observable rate-limiting elementary step, i.e. the conversion of the external aldimine to the pyridoxamine form, being 7.0 x 10(-2)s-1. Aspartate aminotransferase (Lys258----His) thus represents a pyridoxal-5'-phosphate-dependent enzyme with significant catalytic competence without an active site lysine residue. Apparently, covalent binding of the coenzyme, i.e. the internal aldimine linkage, is not essential for the enzymic transamination reaction, and a histidine residue can to some extent substitute for lysine 258 which is assumed to act as proton donor/acceptor in the aldimine-ketimine tautomerization.

Animals↗
PubMed1987

Effect of vitamin B6 on the synthesis and degradation of aspartate aminotransferase in chicken embryo fibroblasts.

The effect of pyridoxal depletion and supplementation on the intracellular level of mitochondrial and cytosolic aspartate aminotransferase in cultured chicken embryo fibroblasts was examined. No apoenzyme was detected in cells grown in the presence of pyridoxal, and the specific activity of total enzyme did not vary profoundly from primary to quaternary cultures. Under pyridoxal depletion, up to 40% apoenzyme was found in tertiary cultures which was entirely due to the mitochondrial isoenzyme. Cytosolic apoenzyme was never detected. Total aspartate aminotransferase relative to total protein was increased 2-fold in secondary cultures; only the mitochondrial isoenzyme contributed to the increased specific activity. The cytosolic isoenzyme decreased steadily and was below the limit of detection in quaternary cultures. The changes are attributed to an increased and decreased synthesis of mitochondrial and cytosolic isoenzyme, respectively. No induction of either isoenzyme was observed after incubating the cells with different hormones and substrates. In secondary cultures, no degradation of mitochondrial isoenzyme could be detected under pyridoxal deficiency or supplementation during 4.4 days, an interpassage duration. The cytosolic aspartate aminotransferase was degraded initially with an apparent half-life of approximately 0.9 day under both sets of conditions. The pronounced stability of mitochondrial aspartate aminotransferase, even though one-third of it was present as apoenzyme, excludes the formation of the apoform to be the rate-limiting step in its degradation. The present results show that pyridoxal affects the synthesis of mitochondrial and cytosolic aspartate aminotransferase, but differently.

Animals↗
PubMed1987

Stereospecific labilization of the C-4' pro-S hydrogen of pyridoxamine 5'-phosphate in aspartate aminotransferase. Activators and inhibitors.

Measurement of the stereospecific release of the pro-S proton from C-4' of enzyme-bound pyridoxamine 5'-phosphate provides an experimental means to probe parts of the active site of aspartate aminotransferase independently of substrate turnover (Tobler, H. P., Christen, P., and Gehring, H. (1986) J. Biol. Chem. 261, 7105-7108). The release of pro-S 3H from enzyme-bound [3H]pyridoxamine 5'-phosphate is 30,000 times faster than from free coenzyme. Enzyme-bound [3H]pyridoxine 5'-phosphate is not detritiated suggesting an essential role of the 4'-amino group. Formation of the unproductive complex of the [3H]pyridoxamine 5'-phosphate-enzyme with aspartate or glutamate results in a 400-fold acceleration of 3H release. In contrast, addition of borohydride or cyanoborohydride immediately stops 3H release. Experiments with a fluorescent reporter group and with differential chemical modifications indicate that the activating effect of aspartate on the release of 3H is accompanied by a shift of the so-called open/closed conformational equilibrium of the enzyme (Kirsch, J.F., Eichele, G., Ford, G. C., Vincent, M.G., Jansonius, J.N., Gehring, H., and Christen, P. (1984) J. Mol. Biol. 174, 497-525) toward the closed conformation; the inhibiting effect of borohydride and cyanoborohydride appears to be accompanied by a shift toward the open conformation. Apparently, at least part of the catalytic apparatus of aspartate aminotransferase becomes fully operative only in the closed conformation of the enzyme.

Animals↗