Evidence for essential histidines in human pituitary glutaminyl cyclase.

Glutaminyl cyclase (QC, EC 2.3.2.5) catalyzes the formation of the pyroglutamyl residue present at the amino terminus of numerous secretory peptides and proteins. Treatment with diethyl pyrocarbonate inactivated recombinant human QC with the apparent modification of three essential histidine residues. Comparisons of the protein sequences of QC from a variety of eukaryotic species show four completely conserved histidine residues. Mutation of each of these residues to glutamine resulted in two mutant enzymes that were inactive (H140Q and H330Q), suggesting a role in catalysis, and two that exhibited increased Km values (H307Q and H319Q), suggesting a role in substrate binding. Consistent with these results is the prediction that QC possesses a zinc aminopeptidase domain in which the four histidines identified here are present in the active site. Mammalian glutaminyl cyclases may, therefore, have structural and catalytic similarities to a family of bacterial zinc aminopeptidases.

Carica papaya glutamine cyclotransferase belongs to a novel plant enzyme subfamily: cloning and characterization of the recombinant enzyme.

A full-length cDNA encoding Carica papaya glutamine cyclotransferase was cloned by RT-PCR on the basis of results from amino acid sequencing of tryptic fragments of the native enzyme. The cDNA of 1036 nucleotides encodes a typical 22-residue signal peptide and a mature protein of 266 residues with a calculated molecular mass of 30,923 Da. Five plant ESTs encoding putative QCs highly homologous to PQC were identified and the numbers and locations of cysteines and N-glycosylation sites are conserved. The plant QC amino acid sequences are very different from the known mammalian QC sequences and no clear homology was observed. The PQC cDNA was expressed in Escherichia coli as either His-tagged PQC, with three different signal peptides and in fusions with thioredoxin, glutathione S-transferase, and (pre-) maltose-binding protein. In all cases, the expressed protein was either undetectable or insoluble. Expression in Pichia pastoris of PQC fused to the alpha-factor leader resulted in low levels of PQC activity. Extracellular expression of PQC in the insect cell/baculovirus system was successful and 15-50 mg/liter of active PQCs with three different secretion signals was expressed and purified. Further, PQC N-terminally fused to a combined secretion signal/His-tag peptide was correctly processed by the host signal peptidase and the His-tag could subsequently be removed with dipeptidyl peptidase I. The expressed products were characterized by activity assays, SDS-PAGE, N-terminal amino acid sequencing, MALDI-TOF mass spectroscopy, and peptide mass fingerprint analysis.

Exploring steric constraints on protein mutations using MAGE/PROBE.

When planning a mutation to test some hypothesis, one crucial question is whether the new side chain is compatible with the existing structure; only if it is compatible can the interpretation of mutational results be straightforward. This paper presents a simple way of using the sensitive geometry of all-atom contacts (including hydrogens) to answer that question. The interactive MAGE/PROBE system lets the biologist explore conformational space for the mutant side chain, with an interactively updated kinemage display of its all-atom contacts to the original structure. The Autobondrot function in PROBE systematically explores that same conformational space, outputting contact scores at each point, which are then contoured and displayed. These procedures are applied here in two types of test cases, with known mutant structures. In ricin A chain, the ability of a neighboring glutamate to rescue activity of an active-site mutant is modeled successfully. In T4 lysozyme, six mutations to Leu are analyzed within the wild-type background structure, and their Autobondrot score maps correctly predict whether or not their surroundings must shift significantly in the actual mutant structures; interactive examination of contacts for the conformations involved explains which clashes are relieved by the motions. These programs are easy to use, are available free for UNIX or Microsoft Windows operating systems, and should be of significant help in choosing good mutation experiments or in understanding puzzling results.

A test strip for diamines in tuna.

This study describes the production of a solid-phase assay (test strip/dipstick test) for putrescine and cadaverine in tuna based on the coupling of an amine oxidase to a peroxidase/dye system. The assay was linear to 75 microM in phosphate buffer, and the minimum detectable concentration was 0.5 microM (< 0.1 ppm), corresponding to 0.01 mg% in spiked extracts. Intra- and interassay precisions were < 20%. Test strips were stable at 4 degrees C for at least 12 months. Lysine, ornithine, and histidine did not react with the assay, and histamine reacted only minimally. Sixteen fish samples were tested by test strip and the standard AOAC protocol, and results were in good agreement.

Evidence for tissue-specific forms of glutaminyl cyclase.

Glutaminyl cyclase (QC) is responsible for the presence of pyroglutamyl residues in many neuroendocrine peptides. An examination of the bovine tissue distribution of QC immunoreactivity, enzyme activity, and mRNA confirmed that QC was abundant in brain and pituitary by all three measures. However, enzymatic activity was considerably more widespread than either immunoreactivity or mRNA, suggesting multiple enzyme forms. Partially purified QC from bovine spleen differed significantly from the known bovine pituitary QC in physical and catalytic properties. We propose that this form of glutaminyl cyclase plays a role in the posttranslational processing of constitutively secreted pGlu-containing proteins.

Substrate and inhibitor specificity of glutamine cyclotransferase (QC).

This paper reports a systematic study of the substrate and inhibitor specificity of papaya latex glutamine cyclotransferase (QC). The results showed that the second amino acid residue in N-terminal glutaminyl peptides significantly accelerated papaya latex QC-catalyzed reactions while the third residue provided no further rate enhancement. Substrate binding was shown to be the main specificity-determining step. Fifteen proline derivatives and dipeptides containing an N-terminal proline were tested and found to inhibit papaya latex QC. This supports our previous molecular modeling study of the QC catalytic pathway which suggested a structure of the reaction intermediates similar to that of L-proline.

Inhibition of cysteine proteinases by Carica papaya cystatin produced in Escherichia coli.

A papaya cystatin (Cst)-encoding cDNA clone was isolated from a papaya leaf cDNA library and the active protein produced in Escherichia coli. The amino-acid sequence reveals a protein of 11,262 Da with over 40% identity to other published plant Cst. Unique features of the papaya Cst include a single Cys residue, variation in the papain-binding region, and the first reported inhibition of papaya proteinase IV by a Cst.

gamma-Glutamyltranspeptidase-catalysed acyl-transfer to the added acceptor does not proceed via the ping-pong mechanism.

Acyl-transfer catalysed by gamma-glutamyltranspeptidase from bovine kidney was studied using gamma-L- and gamma-D-Glu-p-nitroanilide as the donor and GlyGly as the acceptor. The transfer of the gamma-Glu group to GlyGly was shown to be accompanied by transfer of the gamma-Glu group to water (hydrolysis). The results were compared with acyl-transfer catalysed by the representative serine protease, alpha-chymotrypsin. The main difference between the kinetic mechanism of the acyl-transfer reactions catalysed by these enzymes, which contain an active-site serine and form an acyl-enzyme intermediate but belong to different enzyme classes, was found to consist in the role of the enzyme-donor-acceptor complex. This complex is not formed at any acceptor concentrations in the acyl-transfer reactions catalysed by the serine proteases. In contrast, in the gamma-glutamyltranspeptidase-catalysed acyl-transfer the pathway going through the ternary enzyme-donor-acceptor complex formed from the enzyme-acceptor complex becomes the main pathway of the transfer reaction even at moderate acceptor concentrations. As a result, gamma-glutamyltranspeptidase catalysis follows a sequential mechanism with random equilibrium addition of the substrates and ordered release of the products. The second distinction concerns the inhibitory effect of the acceptor. In the case of alpha-chymotrypsin this was the result of true inhibition, i.e. a dead-end formation of the enzyme-acceptor complex. A salt effect caused by the acceptor was the rationale of a similar effect observed in acyl-transfer catalysed by gamma-glutamyltranspeptidase.

Molecular cloning, sequence analysis and expression of human pituitary glutaminyl cyclase.

A cDNA clone for glutaminyl cyclase was isolated from a human pituitary cDNA library and the complete DNA sequence determined. The cDNA clone had 1573 bp and contained an open reading frame of 1086 bases, coding for a protein of 361 amino acids and molecular mass of 40,876 Da. The predicted amino acid sequence of the human cDNA showed 86% sequence identity to the previously reported bovine glutaminyl cyclase sequence. A comparison of the amino acid sequences derived from the human and bovine cDNAs showed that several glycosylation and phosphorylation sites as well as two cysteine residues (Cys139, Cys164) were conserved. The human cDNA was cloned into the Escherichia coli expression vectors pMALc2 and pET19b. Expression of this cDNA in either vector resulted in the production of a glutaminyl cyclase fusion protein which was enzymatically active and reacted with anti-bovine glutaminyl cyclase antisera. Substrate specificity studies with the recombinant enzyme suggested a bias against acidic and tryptophan residues adjacent to the N-terminal glutaminyl residue and a lack of importance of chain length after the second residue.

Steady-state kinetics of glutamine cyclotransferase.

The reaction mechanism of papaya latex glutamine cyclotransferase was studied using pH and temperature dependencies, a proton inventory technique, and molecular modeling. The pH-dependence of the Michaelis-Menten parameters showed that the published pH dependence of the enzyme "activity" was mainly the result of pH-dependent change of the active (unprotonated) substrate concentration. The enzyme activity as such changed very slightly in the pH range between 4.5 and 10. The solvent kinetic isotope effect reflected a change in Vm while the proton inventory was found to be linear with the fractionation factor of the exchangeable proton in the transition state of 0.785. The results were not consistent with an acyl-enzyme mechanism, but rather favored a simple intramolecular cyclization of the glutamine residue to the pyroglutamic acid residue. The mechanism proposed consists of the following main steps: (i) intramolecular nucleophilic attack on the gamma-C = O carbon by the nitrogen of the alpha-amino group, (ii) transfer of a proton from the alpha-amino group to the nitrogen of the amide group, facilitated by an acidic group of the enzyme, and (iii) expulsion of the ammonia-leaving group promoted by this or another acidic enzyme group.

Instability of the ABTS/peroxidase reaction product in biological buffers.

The commonly used horseradish peroxidase dye substrate, 2,2'-azino-bis(3-ethyl-benzthiazoline-6-sulphonic acid) (ABTS), was found to be unstable under certain conditions after reaction with the peroxidase. A survey of several buffers and pH values showed that the oxidized dye was destabilized by raising the pH or by the use of "Good" buffers. It is recommended that use of this dye in a peroxidase-based detection system be confined to acetate buffer at low pH, if possible, and that phosphate or Tris buffers be used if a pH near neutrality is required.

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.

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.

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.

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.

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.

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.

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.