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R Schinzel

Publications and source records attributed to R Schinzel.

65 records · Page 4Linked to original sources
PubMed1994

Dissecting differential binding in the forward and reverse reaction of Escherichia coli maltodextrin phosphorylase using 2-deoxyglucosyl substrates.

Substrate analogs are used in combination with site-directed mutagenesis to probe specific interactions between substrate and enzyme in the forward and reverse direction of the Escherichia coli maltodextrin phosphorylase reaction. In the phosphorolysis (degradation) mode, removal of the 2-OH group of the terminal glucose of the polysaccharide results in a 30-fold reduction of Km while similar changes were of no influence when the same polysaccharide was used for priming the synthesis. Mutation of active site residues Glu637 or Tyr538 does not change apparent affinity of substrates during degradation. In the synthesis mode, 2-deoxyglucose-1-P as substrate causes a 2-fold reduction of the wild-type kcat/Km while for the Y538F mutant a approximately 7-fold reduction is observed. In contrast, the mutation of Glu637 to Asp causes a 10-fold increase in kcat/Km. Therefore, different binding sites for the terminal glucose residue of the oligosaccharide and glucose-1-P exist. Glu637 and Tyr538 are part of the glucose-1-P binding site and do not interact with the terminal glucose residue. A 2-fold increase in rate was observed in both directions using the 2-deoxy derivatives. This confirms the role of intrinsic electronic effects in stabilizing the transition state. Uncompetitive substrate inhibition at high concentrations of maltoheptaose in the phosphorolysis direction is explained by inhibitory binding of the sugar in the synthesis mode.

Amino Acid Sequence↗
PubMed1994

The cognition-enhancing drug tenilsetam is an inhibitor of protein crosslinking by advanced glycosylation.

Non-enzymatic glycosylation of proteins, also called Maillard reaction, which occurs at an accelerated rate in diabetes, can lead to the formation of advanced glycosylation endproducts (AGEs). Tenilsetam (CAS 997: (+/-)-3-(2-thienyl)-2-piperazinone), a cognition-enhancing drug successfully used for treatment of patients suffering from Alzheimer's disease, when included in the Maillard reaction apparently inhibits protein crosslinking by AGEs in vitro. According to the mechanism proposed, Tenilsetam acts via covalent attachment to glycated proteins, thus blocking the reactive sites for further polymerisation reactions. A beneficial effect of Tenilsetam in Alzheimer's disease could come from the interference with AGE-derived crosslinking of amyloid plaques and a decreased inflammatory response by diminished activation of phagocytosing microglia.

Aging↗
PubMed1993

Reaction of imidazole-citrate-deformed glycogen phosphorylase with amino acids.

Incubation of phosphorylase with L-valine in the presence of 0.4 M imidazole citrate results in a time-dependent decrease in the absorption of the enzyme-bound cofactor pyridoxal 5'-phosphate at 333 nm and the generation of a new absorption maximum at 415 nm which appears to be due to an enzyme-bound coenzyme-amino-acid aldimine adduct. Consequently, the enzyme is inactivated to less than 10% of its initial activity. The formation of the adduct of phosphorylase b with L-valine (0.1 M) proceeds with t1/2 approximately 8 min at pH 6.8 and 25 degrees C and is slightly inhibited by AMP. Phosphorylase a reacts five times more slowly than phosphorylase b. The decrease in enzymic activity is linked to the formation of the coenzyme-amino-acid adduct and is not due to resolution of the enzyme. Both the original absorption spectrum and phosphorylase activity are restored by gel filtration in the absence of L-valine and imidazole citrate. Similar reactions occur with other L-amino acids, an exception being L-cysteine which leads to resolution of the enzyme [Shaltiel, S., Hedrick, J. L. & Fischer, E. H. (1966) Biochemistry 5, 2108-2116]. No reaction is observed with D-amino acids or in the absence of imidazole citrate. Pyridoxal-reconstituted phosphorylase rapidly produces with amino acids not only the aldimine adduct but also a species absorbing at 318 nm. Enzyme-bound pyridoxal 5'-phosphate and pyridoxal exhibit a positive CD signal in the region of 333 nm; in contrast, the absorption bands of the coenzyme-amino-acid adducts at 415 nm and 318 nm are optically inactive. Neither pyridoxal-5'-phosphate-reconstituted nor pyridoxal-reconstituted phosphorylase in imidazole citrate catalyses any of the common pyridoxal-5'-phosphate-mediated reactions of amino acids, e.g. transamination, decarboxylation or racemization, thus testifying to the high degree of reaction specificity of phosphorylase.

Amino Acids↗
PubMed1992

Pyridoxal 5'-phosphate as a 31P reporter observing functional changes in the active site of Escherichia coli maltodextrin phosphorylase after site-directed mutagenesis.

Changes in the active site of Escherichia coli maltodextrin phosphorylase created by substituting residues Lys533, Arg534, Tyr538, and Glu637 were monitored in the absence and presence of arsenate as substrate analogue using pyridoxal-P as 31P NMR reporter. The chemical shift of the cofactor phosphate group of wild-type E. coli phosphorylase is pH dependent with an apparent pK of 5.6 and limiting delta values of 0.71 and 3.6 ppm for the low- and high-pH values, respectively. The apparent pK value of 5.6 indicates that the phosphate group of the cofactor is in hydrogen bond linkage to Lys533. In all mutant enzymes in which the enzymatic activity was significantly reduced, effects on the 31P chemical shift pattern of pyridoxal-P were observed. The K533S, R534Q, E637D, and E637Q mutant enzymes show 0.6, 0.01, 0.2, or 0.1% residual activity, and the apparent pK values of the cofactor phosphate transition of E637D and E637Q mutant enzymes are altered. The Y538F mutant enzyme is a remarkable exception, displaying 12% activity and an environment of the cofactor quite similar to that in wild-type enzyme. This finding suggests that Tyr538, although involved in substrate binding and specificity, is not functionally essential. One crucial aspect of catalysis is the close contact of the phosphates of pyridoxal-P and of substrate rendered by a cluster of positively charged amino acids, Lys533, Lys539, and Arg534. The similar apparent pK values of wild-type and K533S mutant phosphorylase suggest that the cofactor phosphate and the hydroxyl group of Ser533 are linked by a hydrogen bond.(ABSTRACT TRUNCATED AT 250 WORDS)

Arsenates↗
PubMed1991

The phosphate recognition site of Escherichia coli maltodextrin phosphorylase.

The role of two positively charged amino acid residues located at the active site of Escherichia coli maltodextrin phosphorylase was investigated by site-directed mutagenesis. Substitution of Lys539 by an arginine caused a 600-fold reduction, substitution of Arg534 by a glutamine caused an even larger 7000-fold reduction of the catalytic rate while substrate binding remained essentially unaffected. Since the Arg534----Gln exchange reduces the catalytic rate near to inactivity and even the conservative Lys534----Arg exchange caused a marked decrease of activity, the central functional role of both positively charged residues in phosphorylase catalysis anticipated by the crystallographic analysis of the corresponding amino acid residues Arg569 and Lys574 in the catalytic site of phosphorylase b was confirmed.

Amino Acid Sequence↗
PubMed1991

Active site lysine promotes catalytic function of pyridoxal 5'-phosphate in alpha-glucan phosphorylases.

Tight contact of pyridoxal 5'-phosphate to the substrate phosphate is considered to be a crucial requirement of the phosphorolytic cleavage of polysaccharides by glycogen phosphorylases. This study demonstrates an essential role of lysine 533, the only charged residue in hydrogen bond distance to the phosphate of pyridoxal 5'-phosphate in Escherichia coli maltodextrin phosphorylase. Substitution of Lys533 by Ser reduced the turnover number 600-fold. Addition of monovalent cations significantly increased activity of the Lys533-Ser mutant up to a factor of 10, whereas the apparent affinity for Pi was decreased up to 80-fold. Although substitution of Lys533 by Gln caused a similar reduction of kcat, the Km values remained unchanged, and no response to small cations was observed. These results suggest a key role of the positive charge contributed by Lys533 in catalysis, most probably in maintaining the electrostatic neutrality of the pyridoxal 5'-phosphate and aligning the close phosphate-phosphate contacts indispensable for the proton transfer mechanism.

Amino Acid Sequence↗
PubMed1990

Escherichia coli maltodextrin phosphorylase: contribution of active site residues glutamate-637 and tyrosine-538 to the phosphorolytic cleavage of alpha-glucans.

The role of Escherichia coli maltodextrin phosphorylase (EC 2.4.1.1) active site residues Glu637 and Tyr538 which line the sugar-phosphate contact region of the enzyme was investigated by site-directed mutagenesis. Substitution of Glu637 by an Asp or Gln residue reduced kcat to approximately 0.2% of wild-type activity, while the Km values were affected to a minor extent. This indicated participation of Glu637 in transition-state binding rather than in ground-state binding. 31P NMR analysis of the ionization state of enzyme-bound pyridoxal phosphate suggested that Glu637 is also involved in modulation of the protonation state of the coenzyme phosphate observed during catalysis. Despite loss of proposed hydrogen-bonded substrate contacts, the Tyr538Phe mutant enzyme retained more than 10% activity; the apparent affinity of all substrates was slightly decreased. Mutations at either site affected the error rate of the enzyme (ratio of hydrolysis/phosphorolysis). Besides a role in substrate binding, the hydrogen-bond network of Tyr538 supports the exclusion of water from the active site.

Amino Acid Sequence↗
PubMed1987

E. coli maltodextrin phosphorylase: primary structure and deletion mapping of the C-terminal site.

The complete 796 residue amino acid sequence of maltodextrin phosphorylase was deduced from the E. coli malP nucleotide sequence. The calculated molecular weight of 90,500, including pyridoxal phosphate, is significantly larger than experimentally determined values. Enzymatically active and inactive mutants following deletion or exchange of up to 8 codons (7 amino acids) at the 3' end (C-terminus) confirm the size of the mature native enzyme and disclose the essential functional or structural role of the highly conserved C-terminal region of phosphorylases.

Amino Acid Sequence↗
PubMed1983

Restriction endonuclease BglI as a tool for in vitro reconstruction and recombination of plasmid and bacteriophage genomes.

The restriction endonuclease BglI produces different individual fragment ends from different cut sites. This property has allowed us to reconstruct efficiently several commonly used plasmid and bacteriophage genomes and a number of recombinant plasmids containing up to seven BglI restriction sites from their constituent BglI fragments. It is demonstrated that in vitro reconstitution from BglI fragments can be used to create, in a simple way, recombinant DNA molecules by recombining in vitro BglI fragments from different mutated or otherwise related genomes. Further applications of the method are discussed.

Bacillus subtilis↗
PubMedDate not supplied

Advanced glycation end-product levels in subtotally nephrectomized rats: beneficial effects of angiotensin II receptor 1 antagonist losartan.

The angiotensin II receptor 1 antagonist losartan (L) inhibited the advanced glycated end-products (AGEs) induced expression of transforming growth factor beta(1) in in vitro experiments performed on renal tubuloepithelial cells. To test the pathophysiological importance of these findings, the possible link between serum AGEs levels and angiotensin system was investigated in the model of normotensive subtotally nephrectomized rats(4/6-NX). Concentration of AGEs in serum of placebo administered 4/6-NX rats (n = 7, 1.09+/-0.09 U/l) increased slightly in comparison with sham-operated healthy controls (CTRL, n = 8, 0.94+/-0.10 U/l, p<0.02) as measured by competitive ELISA. Treatment of 4/6-NX rats with L over 12 weeks ameliorated the rise in serum AGEs concentration (1.00+/-0.12 U/l, n = 15 <0.005) almost to the level observed for CTRL. This effect was further corroborated by the observation, that the impaired renal excretion of AGEs in 4/6-NX-placebo rats (0.07+/-0.02 U/micromol creatinine) was significantly restored by L (0.09+/-0.02 U/micromol creatinine, <0.009) and resembled that of the CTRL (0.10+/-0.03 U/micromol creatinine). Administration of L to 4/6-NX rats significantly improved renal function as evaluated by a smaller rise in serum creatinine and urea concentration. In spite of the improvement in renal function, there were no differences in concentrations of transforming growth factor beta(1) in serum and in urine among the two groups. These effects were independent of blood pressure. Our data give first evidence, that long-term treatment with angiotensin II receptor 1 antagonist may exert salutary effects on AGEs levels in the rat remnant kidney model, probably due to improved renal function.

Angiotensin Receptor Antagonists↗