Analysis of the importance of arginine 102 in neutral endopeptidase (enkephalinase) catalysis.

Neutral endopeptidase 24.11 contains an active site arginine believed to function in substrate binding. This arginine is thought to form an ionic interaction with the COOH-terminal carboxylate of NEP substrates. The functionality of arginine 102 has been investigated by using site-directed mutagenesis to produce mutants in which this residue was converted to a lysine, glycine, glutamine, or glutamate. All of the mutants exhibited essentially full activity as determined with a synthetic peptide amide, glutaryl-Ala-Ala-Phe-4-methoxy-2-naphthylamide. In contrast, activity was detected only with the wild-type enzyme and the lysine mutant using a synthetic substrate containing a free COOH-terminal carboxylate, dansyl-Gly-Trp-Gly. Inhibition studies with the physiologically active peptide substrates substance P, endothelin, and angiotensin I, as well as substance P free acid, [D-Ala2,Leu5]enkephalin, and [D-Ala2,Leu5]enkephalinamide indicated a lack of importance of arginine 102 in substrate binding. With [D-Ala2,Met5]enkephalin and the chemotactic peptide, N-formyl-Met-Leu-Phe, a significant decrease in affinity is observed with the arginine 102 mutants. These results suggest that the contribution of arginine 102 to substrate binding is dependent upon the strength of other subsite interactions. Examination of dipeptides as inhibitors indicates that the nature and orientation of the P'2 residue is important in determining the strength of the interaction of arginine 102 with its substrates.     

Purification and characterization of a human kidney neutral endopeptidase that hydrolyzes succinyl trialanine-4-nitroanilide and biologically active peptides

An enzyme hydrolyzing succinyl trialanine-4-nitroanilide was extracted from human kidney homogenate and purified by means of gel filtration on Sepharose CL-4B, anion-exchange chromatography on DEAE-Sepharose CL-6B and affinity chromatography on carbobenzoxy-L-Ala-L-Ala-D-Ala-polylysine-agarose. The purified enzyme consists of a single peptide, and its molecular weight was estimated to be about 125 000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme cleaved the substrate at the bond between succinyl dialanine and alanine-4-nitroanilide and showed a Km value of 2.1 mM at the optimal pH of 8.0. The activity was increased by Ca2+ and Mg2+, but was inhibited by phosphoramidon and ethylenediaminetetraacetic acid. The enzyme cleaved the oxydized insulin B chain, angiotensinogen tetradecapeptide, angiotensin I, angiotensin II, angiotensin III, [Sar1,Ala8]-angiotensin II, bradykinin, des-Pro2-bradykinin, Leu5-enkephalin, Met 5-enkephalin, [D-Ala2,Met5]-enkephalinamide and [D-Ala2-Met5]-enkephalin, but did not cleave [D-Ala2,D-Leu5]-enkephalin. The bonds on the amino side of the hydrophobic amino acids of the peptides were cleaved by the enzyme.     

Further Characterization of an Enkephalin-Generating Enzyme from Adrenal Medullary Chromaffin Granules

An adrenomedullary protease capable of generating Met5-enkephalin from endogenous precursor(s) has been purified 1,000-fold using affinity chromatography in combination with gel filtration. This trypsin-like enzyme has an apparent molecular weight of 20,000 daltons by gel filtration. The reactivity of the enzyme toward several fluorogenic peptides, Peptides E and F, and the heptapeptides, Met5-enkephalin-Arg6-Phe7 and Met5-enkephalin-Arg6-Arg7, was examined. The two heptapeptides and the fluorogenic compounds were poor substrates for the adrenal enzyme; in contrast, Peptides E and F were cleaved. The low molecular weight products of Peptide F digestion were identified by HPLC as Arg1-Met6-enkephalin, Met5-enkephalin, and Met5-enkephalin-Lys6, while digestion of Peptide E resulted in the production of Leu5-enkephalin and Met5-enkephalin-Arg6-Arg7. [3H]-beta m-Lipotropin was not hydrolyzed by the adrenal enzyme. These results indicate that this adreno-medullary protease is capable of cleaving adrenal opioid peptides at the paired basic sites and thus represents a possible candidate for a proenkephalin-converting enzyme.     

The effects of opiate agonists on growth hormone and prolactin release in rats

Various opioid receptor agonists, including Met5-enkephalin amide, Leu5-enkephalin amide, [D-Ala]2-Met5-enkephalin amide, [D-Ala]2-Leu5-enkephalin amide, morphine sulfate, d-methadone hydrochloride, and l-methadone hydrochloride were administered to adult male rats by subcutaneous injection. All opioid receptor agonists except Leu5-enkephalin amide significantly stimulated growth hormone and prolactin release. Naloxone and naltrexone blocked the hormone stimulatory effects of the opioids and both naloxone and naltrexone, when administered alone, significantly reduced serum growth hormone and prolactin concentrations. The dopaminergic agonist apomorphine, but not the alpha-adrenergic agonist clonidine, blocked opiate stimulation of prolactin. Morphine sulfate caused growth hormone release in rats pretreated with alpha-methyl-p-tryosine, a catecholamine synthesis inhibitor. Cholinergic agonists, physostigmine and pilocarpine, antagonized the growth hormone and prolactin release induced by morphine sulfate. The data suggest that the opiates stimulate prolactin via an interaction with catecholaminergic neurons controlling prolactin release and stimulate growth hormone via a mechanism independent of alpha-adrenergic or general catecholaminergic influence. The mechanism through which cholinergic agonists act to inhibit opiate agonist stimulation of growth hormone is presently unknown.     

Evidence that both arginine 102 and arginine 747 are involved in substrate binding to neutral endopeptidase (EC 3.4.24.11)

Neutral endopeptidase (EC 3.4.24.11, NEP) is a Zn-metallopeptidase involved in the degradation of biologically active peptides, notably the enkephalins and atrial natriuretic peptide. Recently, the structure of the active site of this enzyme has been probed by site-directed mutagenesis, and 4 amino acid residues have been identified, namely 2 histidines (His583 and His587), which act as zinc-binding ligands, a glutamate (Glu584) involved in catalysis, and an arginine residue (Arg102), suggested to participate in substrate binding. Site-directed mutagenesis has now been used to investigate the role of 4 other arginine residues (Arg408, Arg409, Arg659, and Arg747) that have been proposed as possible active site residues and to further analyze the role of Arg102. In each case, the arginine was replaced with a methionine, and both enzymatic activity and the IC50 values of several NEP inhibitors were measured for the mutated enzymes and compared to wild-type enzyme. The results suggest that 2 arginines, Arg102 and Arg747, could both be important for substrate and inhibitor binding. Arg747 seems to be positioned to interact with the carbonyl amide group of the P'1 residue and can be modified when the enzyme is treated with the arginine-specific reagents phenylglyoxal and butanedione. Arg102 could be positioned to interact with the free carboxyl group of a P'2 residue in some substrates and inhibitors and can be modified by phenylglyoxal but not by butanedione. The results could explain the dual dipeptidylcarboxypeptidase and endopeptidase nature of NEP.     

Opioids, Noradrenaline and GTP Analogs Inhibit Cholera Toxin Activated Adenylate Cyclase in Neuroblastoma × Glioma Hybrid Cells

D-Ala2-Met5-enkephalin, morphine, and noradrenaline inhibit the adenylate cyclase in homogenates of neuroblastoma x glioma hybrid cells in a dose-dependent manner even after the enzyme has been preactivated by cholera toxin. Half-maximal inhibition and extent of inhibition are the same with native or cholera toxin-activated enzyme. The inhibition caused by opioids or noradrenaline are antagonized by naloxone or phentolamine, respectively. The effect of D-Ala2-Met5-enkephalin on cholera toxin-activated enzyme is immediate in onset and rapidly reversed by the addition of naloxone. Guanyl-5'-yl-imidodiphosphate stimulates basal activity but inhibits the enzyme activated by cholera toxin or prostaglandin E1. Stimulation occurs at a concentration of 100 microM or above, inhibition even at 0.1 microM. The inhibitory effect of the non-hydrolysable GTP analog is antagonized by GTP. Guanyl-5'-yl-methylenediphosphonate, another nonhydrolysable GTP analog, inhibits basal as well as cholera toxin-stimulated or prostaglandin E1-stimulated adenylate cyclase. Other guanine derivatives such as GDP, GMP, cyclic GMP, guanyl-5'-yl-phosphoric acid amide and guanosine have no effect under the same conditions. The results may be taken as a piece of evidence for two separate guanyl nucleotide-binding sites accompanying the adenylate cyclase in the hybrid cells and mediating, respectively, stimulation and inhibition of the enzyme by hormones.     

Isolation and characterization of a basic carboxypeptidase from human seminal plasma

A carboxypeptidase which cleaves the C-terminal arginine or lysine from peptides was purified by a two-step procedure; gel filtration on Sephacryl S-300 and affinity chromatography on arginine-Sepharose. The activity increased 280% after the first step, indicating the removal of an inhibitor from the crude starting material. The activity in the crude seminal plasma eluted from the Sephacryl S-300 column with an apparent Mr 98,000 and after purification with an Mr 67,000, indicating that it binds to another protein in the crude seminal plasma. When analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, a single band at Mr 53,000 was seen which was converted to two smaller bands (Mr 32,000 and/or 26,000) after reduction. The seminal plasma carboxypeptidase has a neutral pH optimum, is inhibited by o-phenanthroline and by the inhibitor of carboxypeptidase B-type enzymes, 2-mercaptomethyl-3-guanidinoethylthiopropanoic acid, and can be activated by cobalt. The purified enzyme has a high specific activity (67.8 mumol/min/mg) with the ester substrate benzoyl (Bz)-Gly-argininic acid and readily cleaves Bz-Ala-Lys, Bz-Gly-Arg, and Bz-Gly-Lys. It also hydrolyzes biologically active peptides such as bradykinin (Km = 6 microM, kcat = 43 min-1), Arg6-Met5-enkephalin (Km = 103 microM, kcat = 438 min-1), and Lys6-Met5-enkephalin (Km = 848 microM, kcat = 449 min-1). The seminal plasma carboxypeptidase did not cross-react with antiserum to human plasma carboxypeptidase N; other properties distinguish it from the blood plasma enzyme as well as from pancreatic carboxypeptidase B and granular, acid carboxypeptidase H (enkephalin convertase). The carboxypeptidase could be involved in the control of fertility by activating or inactivating peptide hormones in the seminal plasma. In addition it could contribute to the degradation of basic proteins during semen liquefaction.     

Microscale affinity purification of trypsin reduces background peptides in matrix-assisted laser desorption/ionization mass spectrometry of protein digests

The use of trypsin for protein digestion is hampered by its autolysis and low thermostability. Chemical modifications have been employed to stabilize the enzyme. Modified trypsin (e.g. methylated) usually enables performing digestions at elevated temperatures, but it still produces autolytic peptides. In this work, unmodified bovine trypsin was subjected to a microscale affinity chromatography on Arginine Sepharose (ASE) or Benzamidine Sepharose (BSE), which utilized the principle of active-site ligand binding. Trypsin was retained on the sorbents in ammonium bicarbonate as a binding buffer. After washings to remove unbound impurities, the enzyme was eluted by arginine as a free ligand (from ASE) or by diluted hydrochloric acid (from BSE). MALDI-TOF mass spectrometry confirmed removal of large molecular fragments as well as autolytic and other background peptides. Consequently, the purified trypsin was tested for its performance in procedures of in-gel digestion of protein standards and selected urinary proteins from real samples. It has been shown that the affinity purification of trypsin decreases significantly the number of unmatched peptides in peptide mass fingerprints. The presence of arginine in the digestion buffer was found to reduce intensity of autolytic peptides. As a result, the described purification procedure is applicable in a common proteomic routine.     

Experimental attempt to simulate receptor site environment. A 500-MHz 1H nuclear magnetic resonance study of enkephalin amides

The amides of Leu5-enkephalin, Met5-enkephalin, and three analogues, D-Ala2,Leu5-enkephalin, (AcO)Tyr1,Met5-enkephalin, and (AcO)Tyr1,D-Ala2,Met5-enkephalin, have been studied by means of 1H NMR spectroscopy in two different solvent systems: Me2SO-d6 and CDCl3. In the latter solvent the peptides were dissolved as complexes with 18-crown-6-ether, a coronand that binds strongly to the NH3+ groups. The crown ether complexation and the apolar solvent were used to simulate the anionic subsite of the receptor and the hydrophobic environment of the receptor cavity, respectively. The very unusual amide proton chemical shifts and their temperature coefficients suggest the presence of folded conformations in CDCl3 for all peptides, consistent with several models of opioid receptors and with the crystal structure of Leu5-enkephalin. The differences among the proposed cyclic conformations of the five peptides may be correlated, in part, with their different biological activity. All peptides in Me2SO-d6 are characterized by complex mixtures of extended fully solvated conformations.     

Photoreactive enkephalin analogue: [D-Ala2, p-N3-Phe4-Met5]-enkephalin. Synthesis, purification by high performance liquid chromatography and characterization

A photoreactive [D-Ala2, p-N3-Phe4-Met5]enkephalin was synthesized by classical solution peptide synthetic methods. The hydroxysuccinimide ester was used in all the coupling steps in the presence of a weak base, triethylamine. The deprotected enkephalin analogue was purified on high performance liquid chromatography using a Waters, C18 muBondapak reverse phase column and its purity was assessed by thin-layer chromatography. The composition of the analogue was determined and confirmed by elemental analysis and amino acid analysis. Its photoreactivity was demonstrated by the time dependent ultraviolet spectral changes on exposure to light. Competition receptor binding showed that [D-Ala2, p-N3-Phe4-Met5]enkephalin was 4-fold more potent than [D-Ala2, Met5]-enkephalin in competing for binding to the enkephalin binding site. The data presented suggest that this photoreactive enkephalin analogue may be suitable for use in the in situ photoaffinity labeling of the enkephalin receptor.     

Mapping the active site of endothelin-converting enzyme-1 through subsite specificity and mutagenesis studies: a comparison with neprilysin

Endothelin-converting enzyme-1 (ECE-1) is a membrane-bound zinc metallopeptidase that is homologous to neprilysin in amino acid sequence. A major in vivo function of ECE-1 is the generation of endothelin-1, a potent vasoconstrictor, from big endothelin-1. ECE-1 is also potentially involved in the processing or degradation of other peptide hormones. In this study we have used substrates based on the sequence of the COOH-terminal half of big endothelin-1 to examine the subsite specificity of recombinant ECE-1. The big endothelin-1 [16-38] peptides were systematically varied at either position 21 (P(1)) or position 22 (P'(1)) and used in steady-state kinetic analyses of ECE-1. The results indicate that the S(1) pocket of ECE-1 is relatively nonselective, but that the S'(1) subsite of ECE-1 has a preference for large hydrophobic side chains. The peptidyl carboxydipeptidase activity of ECE-1 was also characterized, revealing that substrates with COOH-terminal carboxylates are highly preferred over the cognate amides and esters. A site-directed mutagenesis study was carried out to identify the active-site amino acid residues specifically involved in binding to the COOH-terminal carboxylate of substrates. The data indicate that Arg(133) of ECE-1, which corresponds to Arg(102) of neprilysin that has been identified as an active-site residue of neprilysin involved in binding to the free carboxylate of some substrate peptides, may not play the same role. However, the low activity observed for an ECE-1 Arg(726) mutant is consistent with a role for this arginine residue in the binding of substrates, a role which has been ascribed to arginine residues in both thermolysin (Arg(203)) and neprilysin (Arg(717)).     

The complete amino acid sequence and identification of the active-site arginine peptide of Escherichia coli 2-keto-4-hydroxyglutarate aldolase

The complete amino acid sequence of 2-keto-4-hydroxyglutarate aldolase from Escherichia coli has been established in the following manner. After being reduced with dithiothreitol, the purified aldolase was alkylated with iodoacetamide and subsequently digested with trypsin. The resulting 19 peptide peaks observed by high performance liquid chromatography, which compared with 21 expected tryptic cleavage products, were all isolated, purified, and individually sequenced. Overlap peptides were obtained by a combination of sequencing the N-terminal region of the intact aldolase and by cleaving the intact enzyme with cyanogen bromide followed by subdigestion of the three major cyanogen bromide peptides with either Staphylococcus aureus V8 endoproteinase, endoproteinase Lys C, or trypsin after citraconylation of lysine residues. The primary structure of the molecule was determined to be as follows. (formula; see text) 2-Keto-4-hydroxyglutarate aldolase from E. coli consists of 213 amino acids with a subunit and a trimer molecular weight of 22,286 and 66,858, respectively. No microheterogeneity is observed among the three subunits. The peptide containing the active-site arginine residue (Vlahos, C. J., Ghalambor, M. A., and Dekker, E. E. (1985) J. Biol. Chem. 260, 5480-5485) was also isolated and sequenced; this arginine residue occupies position 49. The Schiff base-forming lysine residue (Vlahos, C. J., and Dekker, E. E. (1986) J. Biol. Chem. 261, 11049-11055) is located at position 133. Whereas the active-site lysine peptide of this aldolase shows 65% homology with the same peptide of 2-keto-3-deoxy-6-phosphogluconate aldolase from Pseudomonas putida, these two proteins in toto show 49% homology.     

The presence of reactive SH groups in the enzymatically active dicyano derivative of creatine kinase

It has been shown that the active dicyano derivative of creatine kinase (ATP:creatine N-phosphotransferase) obtained by cyanolysis of the 5,5'-dithiobis(2-nitrobenzoic acid)-modified and inactivated enzyme contains, as does the native enzyme, two reactive SH groups. Modification of these two SH groups leads to complete inactivation of the dicyano enzyme. Reaction with 4-iodoacetamido-1-naphthol introduces fluorescent labels at these reactive SH groups of the native and the dicyano enzymes. Following tryptic digestion, the respective fluorescent-labelled peptides have been separated by HPLC and the amino acid composition analysis of these peptides has shown that they are consistent with the sequence of the peptide segment containing the active-site SH of Cys-282 of creatine kinase for both the native and the dicyano enzymes, showing that the active SH groups are free in the dicyano enzyme. Upon mild denaturation in 3 M urea, it can be shown that two of the SH groups partially buried in the native enzyme have been cyanylated in the dicyano enzyme. The two reactive SH groups are therefore essential for the activity of creatine kinase and the two cyanylated SH groups are internal groups which probably contributes partially to the stabilization of an active conformation of the enzyme molecule.     

Ribonuclease activity of the peptides with alternating arginine and leucine residues conjugated to tetrathymidilate

RNA cleaving conjugates have been prepared by attachment of oligodeoxyribonucleotide TTTT to peptides containing arginine, leucine, proline and serine residues. The highest activity was displayed by the conjugates containing peptides with alternating arginine and leucine residues (LR)4G-amide. Ribonuclease activity of the conjugates pep-T4 decreases in the order T4-(LR)4G > T4-(LR)2G > T4-(LLRR)2G > T4-(LR)2PRLRG > S2R3-Hmda-T4 > or = R5 double dagger (LR)3. According to CD spectra, the free peptide (LR)4G-amide in water solution at neutral pH and physiological ionic strength has no pronounced secondary structure whereas conjugated to oligonucleotide it acquires a folding similar to alpha-helix.     

Identification of the C-1-phosphate-binding arginine residue of rabbit-muscle aldolase. Isolation of 1,2-cyclohexanedione-labeled peptide by chemisorption chromatography.

The arginine-specific reagent 1,2-cyclohexanedione reacts selectively with the arginine residue of the C-1-phosphate-binding site of aldolase and inactivates the enzyme. The labeled peptide isolated from tryptic digests of inactivated aldolase was found to correspond to the sequence Leu-43 to Arg-56, the residue modified by cyclohexanedione being Arg-55. This peptide was absent form digests of aldolase treated in the same way but protected from inactivation by the presence of substrate, thus correlating modification of Arg-55 with loss of activity. Selective isolation ofthe peptide containing the modified arginine residue was effected by chemisorption chromatography on boric acid gel, a procedure exploiting the specific interaction of matrix-bound boric acid groups with vicinal cis-hxdroxyl groups of cyclohexanedione-modified arginine side chains.     

Selective isolation of lysine-free tryptic peptides delimited by arginine residues: A new tool for proteome analysis

Tryptic digestion of biotinylated Lys-C peptides followed by affinity chromatography allows the selective isolation of lysine-free tryptic peptides delimited by arginine residues (RRnK peptides). In silico analysis revealed that RRnK peptides represent 87% of the whole proteomes and their specific isolation simplifies the complex peptide mixture (5 peptides per protein). The good recoveries and high selectivity obtained in the isolation of RRnK peptides anticipate the applicability of this method in 2DE-free quantitative proteome analyses.     

Degradation of low-molecular-weight opioid peptides by vascular plasma membrane aminopeptidase M

Since both aminopeptidases and angiotensin I-converting enzyme are reported to degrade circulating enkephalins, we have examined the degradation of low-molecular-weight opioid peptides by a vascular plasma membrane-enriched fraction previously shown to contain both angiotensin I-converting enzyme (EC 3.4.15.1) and aminopeptidase M (EC 3.4.11.2). Except for an enkephalin analog resistant to amino-terminal hydrolysis, [D-Ala2]enkephalin, the purified vascular plasma membrane preferentially degraded low-molecular-weight opioids by hydrolysis of the N-terminal Tyr-1--Gly-2 bond. Enkephalin degradation was optimal at pH 7.0 and was inhibited by the aminopeptidase inhibitors amastatin (I50 = 0.08 microM), bestatin (9.0 microM) and puromycin (80 microM). Maximal rates of hydrolysis, calculated per mg plasma membrane protein, were highest for the shorter peptides (18.3, 15.6 and 16.6 nmol/min per mg for Met5-enkephalin, Leu5-enkephalin and Leu5-enkephalin-Arg6, respectively) and decreased with increasing peptide length (0.7 nmol/min per mg for dynorphin (1-13)). No significant hydrolysis of beta- and gamma-endorphin was detected. Km values decreased significantly with increasing peptide length (Km = 72.9 +/- 2.7, 43.6 +/- 4.7 and 21.4 +/- 0.9 microM for Met5-enkephalin, Leu5-enkephalin-Arg6 and Met5-enkephalin-Arg6-Phe7, respectively). However, no further decreases were seen with even larger sequences, i.e., dynorphin(1-13). Other peptides hydrolyzed by the plasma membrane aminopeptidase (angiotensin III, kallidin and hepta(5-11)-substance P) inhibited enkephalin degradation in a competitive manner. Thus, localization, specificity and kinetic data are consistent with identification of aminopeptidase M as a vascular enzyme with the capacity to differentially metabolize low-molecular-weight opioid peptides within the microenvironment of vascular cell surface receptors. Such differential metabolism may play a role in modulating the vascular effects of peripheral opioids.     

Novel mu-selective Met-enkephalinamide analogs with antagonist activity. Synthesis, receptor binding, analgesic properties and conformational studies

Four novel mu-selective peptide antagonists have been synthesized and examined for receptor binding, analgesic agonist and antagonist activity and energy conformational properties. These peptides were designed by analogy to results of molecular modeling of 3-phenyl piperidines which led to incorporating four modified tyrosine residues, m-Tyr, beta-methyl-m-Tyr, N-phenethyl-m-Tyr and alpha, beta-dimethyl-m-Tyr into D-Ala2-Met5-enkephalinamide. Peptides were synthesized by stepwise solution synthesis using an active ester coupling procedure. Receptor binding assays were performed on rat brain homogenates and data were analyzed by a modified version of the program LIGAND. Analgesic agonist and antagonist activity was evaluated by the mouse tail-flick test. Energy-optimized conformations were obtained using a program called Molecule-AIMS. The results demonstrate that relative ratios of in vivo agonist and antagonist potencies in D-Ala2-Met5-enkephalinamides can be modulated by chemical modification of the tyrosine residue. A shift in the phenolic-OH position from para to meta significantly enhances relative antagonist versus agonist activity; addition of a beta-CH3 group to the m-Tyr enhances mu-selectivity and leads to nearly equal agonist/antagonist activity. Energy conformational studies indicate that all analogs with high mu-receptor affinity examined have a common energy accessible B'II 2-3 turn conformation similar to that previously identified for high mu-affinity binding in peptides, lending further support to this candidate conformer. This conformer also has tyrosine side-chain angles which allowed total overlap with the amine and phenolic groups of a known structure of 3-(m-OH phenyl)-piperidine. This structural similarity together with the observation of mixed agonist antagonist activity in both types of opioids confirms the rationale upon which design of these peptides was based.     

Role of aspartate 400, arginine 262, and arginine 401 in the catalytic mechanism of human coproporphyrinogen oxidase

Coproporphyrinogen oxidase (CPO) is the sixth enzyme in the heme biosynthetic pathway, catalyzing two sequential oxidative decarboxylations of propionate moieties on coproporphyrinogen-III forming protoporphyrinogen-IX through a monovinyl intermediate, harderoporphyrinogen. Site-directed mutagenesis studies were carried out on three invariant amino acids, aspartate 400, arginine 262, and arginine 401, to determine residue contribution to substrate binding and/or catalysis by human recombinant CPO. Kinetic analyses were performed on mutant enzymes incubated with three substrates, coproporphyrinogen-III, harderoporphyrinogen, or mesoporphyrinogen-VI, in order to determine catalytic ability to perform the first and/or second oxidative decarboxylation. When Asp400 was mutated to alanine no divinyl product was detected, but the production of a small amount of monovinyl product suggested the K(m) value for coproporphyrinogen-III did not change significantly compared to the wild-type enzyme. Upon mutation of Arg262 to alanine, CPO was again a poor catalyst for the production of a divinyl product, with a catalytic efficiency <0.01% compared to wild-type, including a 15-fold higher K(m) for coproporphyrinogen-III. The efficiency of divinyl product formation for mutant enzyme Arg401Ala was approximately 3% compared to wild-type CPO, with a threefold increase in the K(m) value for coproporphyrinogen-III. These data suggest Asp400, Arg262, and Arg401 are active site amino acids critical for substrate binding and/or catalysis. Possible roles for arginine 262 and 401 include coordination of carboxylate groups of coproporphyrinogen-III, while aspartate 400 may initiate deprotonation of substrate, resulting in an oxidative decarboxylation.     

Escherichia coli twin arginine (Tat) mutant translocases possessing relaxed signal peptide recognition specificities

The twin arginine (Tat) secretion pathway allows the translocation of folded proteins across the cytoplasmic membrane of bacteria. Tat-specific signal peptides contain a characteristic amino acid motif ((S/T)RRXFLK) including two highly conserved consecutive arginine residues that are thought to be involved in the recognition of the signal peptides by the Tat translocase. Here, we have analyzed the specificity of Tat signal peptide recognition by using a genetic approach. Replacement of the two arginine residues in a Tat-specific precursor protein by lysine-glutamine resulted in an export-defective mutant precursor that was no longer accepted by the wild-type translocase. Selection for restored export allowed for the isolation of Tat translocases possessing single mutations in either the amino-terminal domain of TatB or the first cytosolic domain of TatC. The mutant Tat translocases still efficiently accepted the unaltered precursor protein, indicating that the substrate specificity of the translocases was not strictly changed; rather, the translocases showed an increased tolerance toward variations of the amino acids occupying the positions of the twin arginine residues in the consensus motif of a Tat signal peptide.