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R C Bateman

Publications and source records attributed to R C Bateman.

11 recordsLinked to original sources
PubMed1990

N-bromoacetyl-D-leucylglycine. An affinity label for neutral endopeptidase 24.11.

Neutral endopeptidase 24.11 is rapidly inactivated by N-bromoacetyl-D-leucylglycine in a reaction which follows first-order kinetics at pH 8 and 37 degrees C. The concentration dependence of inactivation revealed saturation kinetics with an apparent Ki of 10 mM and kappa inact of 0.4 min-1 at saturating inhibitor concentration. Enzyme can be protected from inactivation by either the substrate Leu5-enkephalin or the competitive inhibitors Phe-Gly or Phe-Ala. Inactivation of enzyme by N-bromo-[14C]acetyl-D-leucylglycine proceeds with the incorporation of a stoichiometric amount of labeled inhibitor. Tryptic digestion of the radioactively labeled enzyme followed by high performance liquid chromatography allowed the isolation of a modified peptide with the sequence T-D-V-H-S-P-G-N-F-R in which histidine (His704) is the modified residue. Site-directed mutagenesis was used to generate a mutant form of the enzyme in which histidine 704 was converted to a glutamine residue. This mutant enzyme retained less than 0.1% of the activity of the native enzyme. These results demonstrate that His704 is at the active site of neutral endopeptidase 24.11 and suggest a catalytic role for this residue.

Affinity Labels↗
PubMed1990

Effect of electron withdrawing substituents on substrate hydrolysis by and inhibition of rat neutral endopeptidase 24.11 (enkephalinase) and thermolysin.

A series of N-acylphenylalanylglycine dipeptides were synthesized and examined as substrates for neutral endopeptidase 24.11 (NEP) and thermolysin. Those N-acyl dipeptides containing an N-acyl group derived from an acid whose pKa is below 3.5 were considerably more reactive with both enzymes than those peptides containing an N-acyl group derived from an acid whose pKa is above 4. The data are interpreted to suggest that electron withdrawal at the scissile bond increases kappa cat for both NEP and thermolysin. The pH dependence for inhibition by the dipeptides Phe-Ala, Phe-Gly, and Leu-Ala showed binding dependent upon the basic form of an enzyme residue with a pKa of 7 for NEP and a pKa of 6 for thermolysin. In the case of thermolysin this pKa was decreased to 5.3 in the enzyme-inhibitor complex. When examined as alternate substrate inhibitors of NEP, N-acyl dipeptides showed three distinct profiles for the dependence of Ki on pH. With N-trifluoroacetyl-Phe-Gly as inhibitor, binding is dependent upon the basic form of an enzyme residue with a pKa value of 6.2. N-methoxyacetyl-Phe-Gly inhibition appears pH independent, while N-acetyl-Phe-Gly inhibition is dependent upon the acidic form of an enzyme residue with a pKa of approximately 7. All inhibitions of thermolysin by N-acyl dipeptides exhibit a dependence on the acidic form of an enzyme residue with a pKa of 5.3 to 5.8. These results suggest that with NEP, binding interactions at the active site involve one or more histidine residues while with thermolysin binding involves an active site glutamic acid residue.

Acylation↗
PubMed1989

Identification of the active-site arginine in rat neutral endopeptidase 24.11 (enkephalinase) as arginine 102 and analysis of a glutamine 102 mutant.

Neutral endopeptidase 24.11 contains an active-site arginine residue involved in binding the free carboxylate of substrate peptides and inhibitors. This arginine reacts rapidly with [14C]phenylglyoxal, and its reaction is selectively blocked by the presence of either the substrate Met5-enkephalin, the competitive inhibitor phenylalanylalanine, or the transition state analog phosphoramidon. The phenylglyoxal-modified peptide was isolated by a procedure involving limited digestion by trypsin, separation of the tryptic peptides by high pressure liquid chromatography (HPLC), further digestion of the modified peptide by pepsin, and a final purification by HPLC. By this procedure arginine 102 was identified as the active-site arginine. Verification of this finding came from the use of site-directed mutagenesis in which this arginine was replaced by glutamine. Both the mutant and wild-type enzyme reacted equally well with an amide containing substrate, glutaryl-Ala-Ala-Phe-4-methoxy-2-naphthylamide. However, reaction of the mutant enzyme with a substrate containing a free COOH-terminal carboxylate, 5-dimethylaminonaphthalene-1-sulfonyl-D-Ala-Gly-(NO2)Phe-Gly, was barely detectable with the mutant enzyme. Similarly the mutant enzyme showed a loss of selectivity in inhibition by D-Ala2-Met5-enkephalin compared to the corresponding amide but exhibited no difference in the maximal velocity for hydrolysis of D-Ala2-Met5-enkephalin and its amide.

Amino Acid Sequence↗
PubMed1989

A spectrophotometric assay for glutaminyl-peptide cyclizing enzymes.

Current assays for the glutaminyl-peptide cyclizing enzyme, glutaminyl cyclase, have several shortcomings. In this report a rapid spectrophotometric assay for cyclization of glutaminyl-peptides to pyroglutamyl-peptides by glutaminyl cyclase is described which overcomes many of these shortcomings. This coupled assay utilizes glutamate dehydrogenase, alpha-ketoglutarate and NADH to measure the ammonia released during cyclization of the dipeptide substrate Gln-Gln. Glutaminyl cyclase from bovine pituitary, partially purified by ion exchange chromatography, exhibits a Km for this substrate of 0.6 mM and a Vmax of 9.6 nmol/min/mg protein. These values are comparable to ones previously reported using other glutaminyl-peptide substrates and either radioimmunoassay or high-performance liquid chromatography to measure glutaminyl cyclase activity.

Acyltransferases↗
PubMed1987

Evidence for an essential histidine in neutral endopeptidase 24.11.

Rat kidney neutral endopeptidase 24.11, "enkephalinase", was rapidly inactivated by diethyl pyrocarbonate under mildly acidic conditions. The pH dependence of inactivation revealed the modification of an essential residue with a pKa of 6.1. The reaction of the unprotonated group with diethyl pyrocarbonate exhibited a second-order rate constant of 11.6 M-1 s-1 and was accompanied by an increase in absorbance at 240 nm. Treatment of the inactivated enzyme with 50 mM hydroxylamine completely restored enzyme activity. These findings indicate histidine modification by diethyl pyrocarbonate. Comparison of the rate of inactivation with the increase in absorbance at 240 nm revealed a single histidine residue essential for catalysis. The presence of this histidine at the active site was indicated by (a) the protection of enzyme from inactivation provided by substrate and (b) the protection by the specific inhibitor phosphoramidon of one histidine residue from modification as determined spectrally. The dependence of the kinetic parameter Vmax/Km upon pH revealed two essential residues with pKa values of 5.9 and 7.3. It is proposed that the residue having a kinetic pKa of 5.9 is the histidine modified by diethyl pyrocarbonate and that this residue participates in general acid/base catalysis during substrate hydrolysis by neutral endopeptidase 24.11.

Animals↗
PubMed1986

Purification and characterization of a peptidyl glycine monooxygenase from porcine pituitary.

A peptide alpha-amidating enzyme was purified to apparent homogeneity from porcine pituitary. This enzyme is a glycoprotein with a mol wt of 64,000, a metal prosthetic group, and a dependence upon ascorbate and molecular oxygen. The purified enzyme has a strong preference for peptides ending in glycine. It also catalyzes the oxidation of valylglycine bonds more rapidly than prolylglycine bonds, and demonstrates a primary isotope effect greater than 5 when the alpha-hydrogens of glycine are replaced by deuterium. Kinetic analysis is consistent with a ping-pong or double displacement catalytic mechanism in which both the peptide substrate and ascorbate are competitive inhibitors with respect to each other. With respect to its kinetic properties, catalytic mechanism, and cofactor requirements, the purified amidating enzyme is very similar to dopamine beta-hydroxylase, a finding which supports the previous suggestion that the peptide alpha-amidating enzyme be classified as a peptidyl glycine monooxygenase.

Animals↗
PubMed1985

Nonenzymatic peptide alpha-amidation. Implications for a novel enzyme mechanism.

An abiotic system is described which chemically catalyzes the formation of less than Glu-His-Pro-NH2 (thyrotropin-releasing hormone) from less than Glu-His-Pro-amino acid in the presence of copper, ascorbate, and molecular oxygen. Evidence is presented to support the participation of hydroxyl and carbon radicals as reaction intermediates in the production of a peptide amide and an aldehyde or ketone. The characteristics of this model system closely mimic the characteristics of enzymatic peptide amidation, and an oxidative, free-radical mechanism for enzymatic peptide amidation is proposed as an alternative to the mechanism for enzymatic amidation offered by Bradbury et al. (Bradbury, A. F., Finnie, M. D. A., and Smyth, D. G. (1982) Nature 298, 686-688).

Amides↗
PubMed1985

A rapid, sensitive assay for glycine-directed amidating enzymes.

Current assays for glycine-directed, peptide-amidating enzymes have several shortcomings. In this report, we describe a rapid, sensitive microassay for amidating activity which overcomes these disadvantages. Tissue homogenates are incubated in the presence of D-Tyr-Val-Gly-OH and the product, D-Tyr-Val-NH2, is measured by radioimmunoassay using an antiserum with an affinity for the product, D-Tyr-Val-NH2 (Kaff 2 X 10(8) L/M), 4 orders of magnitude higher than for the substrate, D-Tyr-Val-Gly-OH (Kaff 4 X 10(4) L/M), and 3 orders of magnitude higher than for the deamidated product D-Tyr-Val-OH (Kaff 8 X 10(5) L/M). Addition of N-ethylmaleimide (0.5 mM) to the enzyme incubates prevents the degradation of D-Tyr-Val-NH2 and permits the measurement of enzymatic activity in crude homogenates. This assay is sensitive enough to permit the measurement of amidating activity in crude rat brain homogenates containing as little as 4-8 micrograms protein.

Animals↗