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C M Metzler

Publications and source records attributed to C M Metzler.

At least 19 recordsLinked to original sources
PubMed1997

Refinement and comparisons of the crystal structures of pig cytosolic aspartate aminotransferase and its complex with 2-methylaspartate.

Two high resolution crystal structures of cytosolic aspartate aminotransferase from pig heart provide additional insights into the stereochemical mechanism for ligand-induced conformational changes in this enzyme. Structures of the homodimeric native structure and its complex with the substrate analog 2-methylaspartate have been refined, respectively, with 1.74-A x-ray diffraction data to an R value of 0.170, and with 1.6-A data to an R value of 0.173. In the presence of 2-methylaspartate, one of the subunits (subunit 1) shows a ligand-induced conformational change that involves a large movement of the small domain (residues 12-49 and 327-412) to produce a "closed" conformation. No such transition is observed in the other subunit (subunit 2), because crystal lattice contacts lock it in an "open" conformation like that adopted by subunit 1 in the absence of substrate. By comparing the open and closed forms of cAspAT, we propose a stereochemical mechanism for the open-to-closed transition that involves the electrostatic neutralization of two active site arginine residues by the negative charges of the incoming substrate, a large change in the backbone (phi,psi) conformational angles of two key glycine residues, and the entropy-driven burial of a stretch of hydrophobic residues on the N-terminal helix. The calculated free energy for the burial of this "hydrophobic plug" appears to be sufficient to serve as the driving force for domain closure.

Acetic Acid↗
PubMed1994

NMR studies of 1H resonances in the 10-18-ppm range for cytosolic aspartate aminotransferase.

Continuing a previous investigation (Kintanar, A., Metzler, C. M., Metzler, D. E., and Scott, R. D. (1991) J. Biol. Chem. 266, 17222-17229), we have recorded 1H NMR spectra at 500 MHz in the 10-18-ppm range for the 93-kDa porcine cytosolic aspartate aminotransferase and for four specific mutant forms of the enzyme in which histidine 68 has been replaced by lysine or histidine 143, 189, or 193 has been replaced by glutamine. We have correlated resonances for apoenzyme, pyridoxamine and pyridoxal phosphate forms, and dicarboxylate complexes and have assigned imidazole NH resonances of active site histidines. The chemical shifts of several resonances undergo pH-dependent changes around the pKa of the Schiff base proton at the active site. Other resonances shift upon binding of dicarboxylates or other ligands. Phosphate or carboxylate ions, which can also occupy the site of the substrate's alpha-carboxylate, cause rapid exchange of the Schiff base proton. Although most resonances in the 10-18-ppm range disappear rapidly in D2O, a few are retained for months in the presence of the dicarboxylate inhibitor glutarate. We demonstrate that changes in chemical shifts and in exchange rates are sensitive indicators of electronic interactions of the enzyme with ligands and of conformational change. Nuclear Overhauser effects from NH protons have allowed us to identify resonances of CH protons of the imidazole rings of histidines 143, 189, and 193. Observed and predicted chemical shifts have been compared. We conclude that the net charge on this histidine cluster is zero but that some negative charge from the aspartate 222 carboxylate is donated inductively into the histidine 143 ring. Studies of the related enzyme from Escherichia coli are provided in an accompanying paper (Metzler, D. E., Metzler, C. M., Scott, R. D., Mollova, E. T., Kagamiyama, H., Yano, T., Kuramitsu, S., Hayashi, H., Hirotsu, K., and Miyahara, I. (1994) J. Biol. Chem. 269, 28027-28033). Our approach should be applicable to the study of active sites of a broad range of relatively large proteins.

Amino Acid Sequence↗
PubMed1994

NMR studies of 1H resonances in the 10-18-ppm range for aspartate aminotransferase from Escherichia coli.

We have recorded 500-MHz 1H NMR spectra in the 10-18-ppm range for aspartate aminotransferase from Escherichia coli and for three specific mutant forms. Histidine 143 has been replaced by either alanine or asparagine. In the third mutant, tryptophan 140 has been replaced by phenylalanine. The NMR spectrum of the native enzyme is very similar to that of porcine cytosolic aspartate aminotransferase in the most downfield region. However, the resonances of the proton on the ring nitrogen of the pyridoxal 5'-phosphate (peak A) and on the His-143 imidazole ring (peak B) of the E. coli enzyme are broader and more readily lost at low pH or higher temperatures than those of the porcine enzyme. The possible role of tautomerism in promoting such broadening is discussed. In the histidine mutant proteins, peak A of the pyridoxal 5'-phosphate form is too broad to see under most conditions but is clearly present in the pyridoxamine phosphate form. Peak B is missing in the 2 histidine mutants. Observation of nuclear Overhauser effects further confirms the identity of B as the resonance of HN epsilon 2 of His-143 and that of peak D at approximately 11.8 ppm as HN epsilon 2 of His-189. The mutant spectra also provide insight into electronic interactions between groups in and near the active site which confirm and supplement conclusions drawn from spectra of porcine cAspAT. While no clear loss of a peak was observed for the Trp-140 mutant in its free form, the spectrum of the succinate complex lacked a strong band at 11.26 ppm. This may represent the Trp-140 indole NH proton which has been shifted downfield by binding to a succinate carboxylate group. While our results confirm the basic similarity of cytosolic aspartate aminotransferase and E. coli aspartate aminotransferase 1H NMR spectra, they also point out differences that may be useful in identifying resonances. A large number of mutant proteins have been prepared for the E. coli enzyme. The present results provide essential information for future study of these mutants and for study of NMR spectra of isotopically labeled enzyme.

Alanine↗
PubMed1994

NMR spectroscopy of exchangeable protons of glucoamylase and of complexes with inhibitors in the 9-15-ppm range.

1H-NMR spectra have been recorded for glucoamylases I and II from Aspergillus awamori var. X100 and from A. niger in the 9-15-ppm region. At least 17 distinct peaks, many of them arising from single protons, are observed. These are designated A-Q, A being the furthest downfield. At least 9 of these are lost rapidly by exchange when the enzyme is placed in D2O. Peaks A, B, E and H undergo distinct shifts with pH change in the pH region 3-7. Several others undergo smaller shifts. Small differences are also seen between the enzymes from the two different sources. Binding of the pseudotetrasaccharide inhibitor acarbose leads to a 0.50-ppm downfield shift of peak B, other smaller changes, and retention of two additional protons in D2O. delta-D-gluconolactone induces shifts in peaks E, H, and L. The slow substrate maltitol causes peak A to broaden and shift, peaks J and K to shift and a new or greatly shifted resonance to appear at 15.4 ppm. It disappears as the maltitol is hydrolyzed. Treatment with iodoacetamide or diethyl pyrocarbonate leads to disappearance of peak D at 12.3 ppm. When this peak was irradiated strong nuclear Overhauser effects (NOE) were observed at 8.01 ppm and 7.22 ppm, positions expected for the C epsilon 1 and C delta 2 protons of an uncharged imidazole ring. We identify D as arising from the N epsilon 2 proton of His254 which is uncharged except at the lowest pH values. Other NOE and two-dimensional NOE spectra have provided additional information. Three mutant forms of the A. niger enzyme, in which tryptophan residues have been replaced by phenylalanine, have been examined. Because of shifts induced by changes in ring current and other environmental effects it is hard to make a direct identification of the resonances from the replaced indole NH protons. However, on the basis of a distinct NOE between peaks E and H we have identified these resonances as arising from the indole NH protons of Trp52 and Trp120. Other possible assignments are considered. The NMR spectra of the glucoamylases I, which have a starch binding domain of about 104 residues at the carboxyl terminus, show four sharp resonances in the 9.7-10.6-ppm range that are not present in the glucoamylases II, which lack this domain. These resonances no doubt represent the four indole NH ring protons from Trp543, Trp562, Trp590 and Trp615. Three of these are very sharp suggesting a high mobility of this domain.

Aspergillus↗
PubMed1994

Starch-binding domain of Aspergillus glucoamylase-I. Interaction with beta-cyclodextrin and maltoheptaose.

The characterization is reported of two peptide fragments (SBD106 and SBD122) containing the starch-binding domain (SBD) of Aspergillus sp. glucoamylase I. The starch-binding peptides were produced in Escherichia coli as fusion proteins of the maltose-binding protein (MBP). SBD106 (11.9 kDa) and SBD122 (13.8 kDa) were purified from the factor Xa digest of MBP fusion proteins. The amino acid compositions were similar to those deduced from their amino acid sequences. The interactions of beta-cyclodextrin and maltoheptaose with purified SBD peptides were investigated by UV difference spectroscopy. SBD106 and SBD122 bound specifically beta-cyclodextrin with a dissociation constant (Kd) of 34 microM and 23.5 microM, respectively. Maltoheptaose binding to SBD106 and SBD122 was weaker than that of beta-cyclodextrin; dissociation constants were 0.57 and 0.50 mM, respectively. The results indicate that the intramolecular disulfide bonding is not required for the domain functioning and that O-glycosylation is not critical for the functioning of the starch-binding domain, but may affect its conformation and dynamics.

Amino Acid Sequence↗
PubMed1994

Acute effects of increasing doses of nicorandil on renal function in man.

The effects of nicorandil, a nicotinamide derived vasodilator combining nitrate and potassium channel opener actions, on kidney function have not been determined. This study investigated changes in renal blood flow and glomerular filtration rate as estimated using simultaneous 131I-iodohippurate and 125I-iothalamate plasma clearances. Forty-two healthy subjects in sodium balance received placebo and 2.5 mg (n = 8), 5 mg (n = 9), 10 mg (n = 8), 20 mg (n = 8) or 30 mg (n = 9) nicorandil orally. Peak nicorandil plasma concentrations occurred in the first hour. Nicorandil produced dose related decreases in blood pressure with maximum reductions (mean +/- standard error of the mean) after 30 mg of -6 +/- 1 mmHg systolic and -8 +/- 2 mmHg diastolic. Renal blood flow averaged 655 +/- 28 ml/minute/1.73 m2 after placebo. Renal blood flow changed 10 +/- 11% after 2.5 mg, -6 +/- 8% after 5 mg, -12 +/- 11% after 10 mg, -11 +/- 5% after 20 mg, and 8 +/- 6% after 30 mg, however, these changes did not reach statistical significance. Glomerular filtration rate averaged 113 +/- 3 ml/minute/1.73 m2 and was unaltered after nicorandil. Nicorandil had no effect on filtration fraction but fractional excretion of sodium tended to decrease with dose. These dose-related effects of nicorandil are consistent with other mixed vasodilators. At therapeutic doses, renal perfusion and function are preserved despite reductions in systemic blood pressure by nicorandil.

Adolescent↗
PubMed1993

Continuous intravenous dosing with ciprostene using a portable pump in ambulatory patients.

Parenteral compounds present special drug delivery challenges. This open-label study evaluated a portable infusion pump as a means to deliver intravenous ciprostene, a stable prostacyclin analog. Ten patients with peripheral vascular disease and claudication received ciprostene (titrated to 120 ng/kg/min) infused over 8 hours 1 day per week for 4 consecutive weeks. Patients successfully maintained the pump strapped to the waist. The mean +/- standard deviation delivery error, with volumes of 6 to 10 mL over 8 hours, was -0.895 +/- 3.177%. Accordingly, the pump performed well with a potent drug under these clinical conditions. Headache, flushing, and infusion site irritation during infusion were the most frequent side effects. Blood pressure remained unchanged during infusion; however, heart rate increased significantly (P < .05, maximum increase was 13.9 +/- 2.1 beats per minute [mean +/- standard error of the mean]. Mean (+/- standard error of the mean) relative claudication times on treadmill remained unchanged; however, absolute claudication times increased (P < .05) from 6.6 +/- 1.8 to 10.0 +/- 2.2 minutes. Ciprostene inhibited adenosine diphosphate-induced platelet aggregation by 56.0 +/- 12.7% (mean +/- standard error of the mean). Mean template bleeding times and plasma concentrations of platelet-specific proteins (beta-thromboglobulin, platelet factor 4) did not change.

Adult↗
PubMed1993

The pharmacokinetics and haemodynamic effects of continuous nicorandil infusion in healthy volunteers.

We have studied the pharmacokinetics and haemodynamic effects of nicorandil after a 12-h infusion. Nicorandil is a mixed vasodilator combining the actions of a nitrate and a potassium channel opener. Nicorandil was infused for 12 h in 21 healthy volunteers at rates of 0.05, 0.10, and 0.20 microgram.kg-1.min-1 using a placebo controlled, crossover design. Systemic blood pressure, heart rate, electrocardiographic (ECG) intervals, and cardiac output (impedance cardiography) were measured supine and standing. Dose-related, steady-state plasma nicorandil concentrations occurred within 3 to 4 h. Nicorandil's pharmacokinetics were linear with dose. Four 0.20 microgram.kg-1.min-1 nicorandil infusions were terminated early primarily because of moderate or severe headaches. There were no safety concerns (ECG intervals, laboratory assays). Blood pressure fell versus placebo only in the standing position and heart rate increased slightly (not significant). That is, standing blood pressure in the 6 to 12 h interval fell from baseline 8.0*/6.8, 1.6/5.1, and 9.8*/7.9* mmHg (systolic/diastolic, * = P < 0.05 versus placebo) at 0.05, 0.10, and 0.20 micrograms.kg-1.min-1 respectively. Cardiac output increased slightly above placebo at lower doses. Haemodynamic changes correlated poorly with plasma nicorandil concentrations. Similar total doses were less well-tolerated when extended over 12 h. We saw no evidence of pharmacodynamic tolerance to nicorandil within 12 h.

Adolescent↗
PubMed1992

Pharmacokinetics and pharmacodynamics of oral diazepam: effect of dose, plasma concentration, and time.

Eleven healthy subjects received single oral doses of placebo, 2 mg diazepam, 5 mg diazepam, and 10 mg diazepam in a randomized four-way crossover study. Plasma diazepam levels, the Digit Symbol Substitution Test (DSST), and fraction of total electroencephalographic (EEG) amplitude falling in the sigma plus beta (13 to 31 Hz) frequency range were determined during the 12 hours after drug administration. Peak plasma diazepam concentration and area under the 12-hour curve were proportional to dose; time of peak was independent of dose. Baseline percentage of EEG amplitude falling in the 13 to 31 Hz range averaged 15.7% and did not differ among the four trials. The percentage of EEG amplitude falling in the 13 to 31 Hz range did not change over baseline with placebo or 2 mg diazepam but was increased 1/4 to 2 1/2 hours after 5 mg diazepam, (maximum, +7.3%) and 3/4 to 12 hours after 10 mg diazepam (maximum, +15.2%). The increase in the percentage of EEG amplitude falling in the 13 to 31 Hz range was highly correlated with plasma diazepam concentration. DSST scores for placebo and 2 mg diazepam were nearly identical. DSST decrements with 5 and 10 mg diazepam paralleled and were correlated with the changes in the percentage of EEG amplitude falling in the 13 to 31 Hz range and with plasma diazepam levels. Thus the EEG analysis provides objective quantitation of benzodiazepine central nervous system effects, in turn reflecting plasma levels and other clinical measures.

Administration, Oral↗
PubMed1991

NMR observation of exchangeable protons of pyridoxal phosphate and histidine residues in cytosolic aspartate aminotransferase.

Observation of the 93-kDa cytosolic aspartate aminotransferase by 500-MHz 1H NMR spectroscopy in H2O has revealed a series of resonances in the 10-18 ppm range arising from exchangeable protons. One of these (peak A) has been assigned to the proton bound to the ring nitrogen of the coenzyme pyridoxal 5'-phosphate. A second (peak B) is assigned to H143 which participates in a chain of hydrogen bonds that includes also the coenzyme-bound proton. There is a mutual nuclear Overhauser effect between these two resonances. Peaks A and B respond to changes in pH and to interaction of the enzyme with coenzyme derivatives and inhibitors. Peak A moves from 15.4 to 17.4 ppm as the pH is lowered, while peak B moves in the opposite direction from 14.7 to 13.7 ppm, both with an apparent pKa of 6.15. This pKa is associated with deprotonation of the imine nitrogen at the Schiff base linkage of the coenzyme with K258 of the enzyme. In spectra of enzyme containing pyridoxamine 5'-phosphate, peak A is observed at 16.5 ppm and peak B is at 13.9 ppm over a broad pH range. Peaks A and B are found at 17.8 and 14.0 ppm, respectively, for the enzyme complex with glutarate. When alpha-methylaspartate is added to the enzyme several new resonances appear in the spectrum, which are attributed to formation of the external aldimine. The position of peak A in spectra of various forms of the enzyme is interpreted to reflect the electronic distribution in the coenzyme ring. Several other peaks in this region of the spectrum also are sensitive to changes in pH or the addition of inhibitors. Some possible assignments of these resonances are discussed.

Aspartate Aminotransferases↗
PubMed1991

NMR spectra of exchangeable protons of pyridoxal phosphate-dependent enzymes.

We have recorded 1H NMR spectra in H2O for exchangeable protons of four pyridoxal phosphate-dependent enzymes: D-serine dehydratase, aspartate aminotransferase, tryptophan: indole-lyase and glutamate decarboxylase. The molecular masses range from 48-250 kDa. In every case there are downfield peaks which are lost when the apoenzyme is formed. In most cases some peaks shift in response to interactions with substrates and inhibitors and with changes in pH. We associate one downfield resonance with the proton on the ring nitrogen of the coenzyme and others with imidazole groups that interact with coenzyme or substrates. The chemical shift for the coenzyme-bound proton differs for free enzyme, substrate Schiff base or quinonoid forms.

Animals↗
PubMed1991

Equilibria and absorption spectra of tryptophanase.

Tryptophanase (tryptophan: indole-lyase) from Escherichia coli has been isolated in the holoenzyme form and its absorption spectra and acid-base chemistry have been reevaluated. Apoenzyme has been prepared by dialysis against sodium phosphate and L-alanine and molar absorptivities of the coenzyme bands have been estimated by readdition of pyridoxal 5'-phosphate. The spectrophotometric titration curve, whose midpoint is at pH 7.6 in 0.1 M potassium phosphate buffers, indicates some degree of cooperativity in dissociation of a pair of protons. Resolution of the computed spectra of individual ionic forms of the enzyme with lognormal distribution curves shows that band shapes are similar to those of model Schiff bases and of aspartate aminotransferase. Using molar areas from the latter we estimated amounts of individual tautomeric species. In addition to ketoenamine and enolimine or covalent adduct the high pH form also appears to contain approximately 18% of a species with a dipolar ionic ring (protonated on the ring nitrogen and with phenolate -O-). We suggest that this may be the catalytically active form of the coenzyme in tryptophanase. The equilibrium between tryptophanase and L-alanine has also been reevaluated.

Escherichia coli↗
PubMed1991

Sample sizes for bioequivalence studies.

In recent years a number of decision rules, based on sound statistical principles, have been proposed for deciding if a test formulation is bioequivalent to a reference formulation. The decision rule based on confidence intervals has been accepted by regulatory agencies, at least by the Food and Drug Administration of the United States. A useful property of this decision rule is that the regulatory agency need not require a certain sample size, since the level of protection against wrongly deciding bioequivalence is set by the choice of the alpha level used to compute the confidence intervals. The manufacturer claiming bioequivalence is concerned about sample size, for sample size determines the probability of falsely deciding non-bioequivalence when the test formulation does indeed have an acceptable relative bioavailability. Curves of probability of rejecting bioequivalence have been computed for error coefficient of variation of 10, 20 and 30 per cent, for relative bioavailability from 70 to 130 per cent, and for protection levels of 90 and 95 per cent. These curves can be used for choosing the sample size for a bioequivalence study.

Bayes Theorem↗
PubMed1990

Lack of effect of fluoride on reproductive performance and development in Shetland sheepdogs.

A 10-y history of high rates of perinatal deaths and congenital anomalies in dogs in a Shetland Sheepdog kennel prompted investigations into the cause(s). Skeletal fluorosis (mottled teeth in pups, bony exostoses in adults) followed use of a commercial dog food later found to contain 460 ppm fluoride (F) from rock phosphate added as a mineral source. The effects of the water source and fluoride in dog food on female reproduction and teratogenicity were investigated in a 2-y, 2-way factorial study in the kennel. Twenty adult shelties of proven fertility were brought into the kennel and randomized into 4 treatment groups, each consisting of 4 females and 1 male. Treatments were: Group A, high F (460 ppm) dog food, well water; Group B, high F dog food, distilled water; Group C, low F (55ppm) dog food, well water; Group D, low F dog food, distilled water. Only Group C produced as many pups as expected. Overall the missed pregnancy rate (44%) in study bitches and perinatal death rate (50%) in 48 study pups paralleled the problems in shelties resident in the kennel. Higher rates for both variables in the high F and the distilled water groups seemed likely due to chance. The only major anomaly was in Group D; minor anomalies appeared in all groups. Although 460 ppm F in dog food did produce bony exostoses, we did not find convincing evidence that it adversely affected reproduction in shelties. The cause of the reproductive problems was apparently not the dog food, water, foliage, genetic factors, or infectious disease and currently remains undetermined.

Abnormalities, Drug-Induced↗
PubMed1989

Reactions of phosphonate analogs of pyridoxal phosphate with apo-aspartate aminotransferase.

We have investigated reactions of the 5-phosphonoethyl and 5-phosphonoethenyl analogs of pyridoxal 5'-phosphate in the coenzyme site of cytosolic aspartate aminotransferase. Acid dissociation constants and equilibrium constants for hydration and for tautomerization have been evaluated for these compounds. In confirmation of previous results, both compounds are partially active. They bind to apoenzyme well and undergo conversion in the presence of glutamate to amine forms which show induced circular dichroism comparable to that of native enzyme. A normal "external" Schiff base is evidently formed with 2-methylaspartate, but the amounts of quinonoid intermediate formed with erythro-3-hydroxyaspartate are less than those formed with pyridoxal phosphate. The pKa of the imine group of the enzyme reconstituted with the phosphonoethyl analog is more than two units lower than that in the native enzyme. Binding of the dicarboxylates glutarate, 2-oxoglutarate, and succinate shifts the pKa upward. The absorption spectra of the resulting complexes indicate the existence of at least three low pH species. A shift of 2.3 to 2.9 ppm to a lower frequency was observed for the 31P NMR signal upon binding of these dicarboxylates or of 2-methylaspartate. Enzyme containing the analogs crystallizes. Polarized absorption spectra suggest that the coenzyme has an orientation similar to that of pyridoxal phosphate in the native enzyme.

Amines↗
PubMed1989

The effects of cefmetazole and latamoxef on platelet function in healthy human volunteers.

This study has compared the effects of normal saline (placebo), cefmetazole, and latamoxef (moxalactam) on platelet function in healthy human volunteers. Twenty-nine volunteers were randomized to receive placebo (n = 10), cefmetazole 2 g (n = 9) or latamoxef 2 g (n = 10) intravenously every 6 h for six days. Under double-blind conditions template bleeding time and ex-vivo adenosine diphosphate (ADP) induced platelet aggregation were measured before drug dosing, after the first, ninth, and last drug doses and one and three days after the last drug dose. Latamoxef caused progressive impairment of platelet function with statistically significant increases in the mean bleeding time to 7.4 +/- 3.7 min (P = 0.02) and the amount of ADP (0.2 mM) required to induce 50% platelet aggregation to 77.3 +/- 91.1 microliters (P = 0.03) on day 6 of treatment compared with values before drug administration (4.2 +/- 0.7 min; 3.7 +/- 22 microliters, respectively). These changes were reversible following discontinuation of drug dosing. Cefmetazole and placebo had no significant effects on either measure of platelet function at any time during the study.

Adenosine Diphosphate↗