Expression of the acidic stretch of nardilysin as a functional binding domain.

Kinetic evidence suggests an acidic region in nardilysin binds polyamines and acts as a regulatory domain. The binding of approximately 5 mol of spermine/mol of nardilysin was demonstrated. The binding curve was sigmoidal exhibiting an IC(50) of approximately 118 microM and a Hill coefficient of 1.8. Spermine diminished the tryptophan fluorescence of the enzyme and increased its sensitivity to protease V8. The acidic stretch from mouse and human nardilysin were expressed as glutathione transferase fusion proteins. All fusion proteins bound spermine with an IC(50) of 40 to 110 microM. The mouse fusion protein bound approximately 7 mol of spermine exhibiting a sigmoidal binding curve and a Hill coefficient of 1.4. The human acidic stretch, containing fewer acidic residues, bound approximately 5 mol of spermine/mol with a hyperbolic binding curve. Chimeric fusion proteins containing the N-terminus of the mouse acidic region fused to the C-terminus of the human acidic region bound approximately 10 mol of spermine, while the opposite chimera bound approximately 4 mol of spermine/mol. The N-terminal region of the mouse acidic domain binds 3--4 mol spermine/mol exhibiting a Hill coefficient of 1.4, while the same region from human nardilysin binds 1 mol of spermine/mol. Spermine enhanced the sensitivity of the mouse acidic domain, but not the human acidic domain, to protease V8. Together the data support a model where the acidic stretch of nardilysin functions as an autonomous domain.

Studies on the subsite specificity of rat nardilysin (N-arginine dibasic convertase).

The subsite specificity of rat nardilysin was investigated using fluorogenic substrates of the type 2-aminobenzoyl-GGX(1)X(2)RKX(3)GQ-ethylenediamine-2,4- dinitrophenyl, where P(2), P(2)', and P(3) residues were varied. (The nomenclature of Schechter and Berger (Schechter, I., and Berger, A. (1967) Biochem. Biophys. Res. Commun. 27, 157-162) is used where cleavage of a peptide occurs between the P(1) and P(1)' residues, and adjacent residues are designated P(2), P(3), P(2)', P(3)', etc.) There was little effect on K(m) among different residues at any of these positions. In contrast, residues at each position affected k(cat), with P(2) residues having the greatest effect. The S(3), S(2), and S(2)' subsites differed in their amino acid preference. Tryptophan and serine, which produced poor substrates at the P(2) position, were among the best P(2)' residues. The specificity at P(3) was generally opposite that of P(2). Residues at P(2), and to a lesser extent at P(3), influenced the cleavage site. At the P(2) position, His, Phe, Tyr, Asn, or Trp produced cleavage at the amino side of the first basic residue. In contrast, a P(2) Ile or Val produced cleavage between the dibasic pair. Other residues produced intermediate effects. The pH dependence for substrate binding showed that the enzyme prefers to bind a protonated histidine. A comparison of the effect of arginine or lysine at the P(1)' or P(1) position showed that there is a tendency to cleave on the amino side of arginine and that this cleavage produces the highest k(cat) values.

New fluorogenic substrates for N-arginine dibasic convertase.

N-Arginine dibasic (NRD) convertase is a recently described peptidase capable of selectively cleaving peptides between paired basic residues. The characterization of this unique peptidase has been hindered by the fact that no facile assay procedure has been available. Here we report the development of a rapid and sensitive assay for NRD convertase, based on the utilization of two new internally quenched fluorogenic peptides: Abz-GGFLRRVGQ-EDDnp and Abz-GGFLRRIQ-EDDnp. These peptides contain the fluorescent 2-aminobenzoyl moiety that is quenched in the intact peptide by a 2, 4-dinitrophenyl moiety. Cleavage by NRD convertase at the Arg-Arg sequence results in an increase of fluorescence. NRD convertase cleaves these peptides efficiently and with high specificity as observed by both HPLC and fluorescence spectroscopy. The rate of hydrolysis of the fluorogenic substrates is proportional to enzyme concentration, and obeys Michaelis-Menten kinetics. The kinetic parameters for the fluorescent peptides (Km values of approximately 1.0 microM, and Vmax values of approximately 1 microM/(min. mg) are similar to those obtained with peptide hormones as substrates.

Kinetic analysis of spermine binding to NRD convertase.

N-arginine dibasic convertase cleaves polypeptides between paired basic residues containing the sequence Arg-Arg or Arg-Lys. The enzyme contains a large anionic domain, which in the rat enzyme consists of 57 acidic residues out of a stretch of 76 amino acids. Polyamines modulate the activity of the enzyme presumably by binding at the anionic domain (Csuhai et al. (1995) Biochemistry 34, 12411-12419). In this study a kinetic analysis of the effect of salts and amines, particularly the polyamine spermine, on the rat enzyme was studied. Simple salts were inhibitory with no apparent specificity for the anion or cation. Inhibition resulted in an increased Km and a decreased Vmax. Evidence that amines bind to an anionic domain was obtained by the finding that N,N-bis [2-hydroxyethyl]-2-aminoethanesulfonic acid, which is structurally related to the inhibitory amine triethanolamine, is noninhibitory. Inhibition exhibited a complex dependence on spermine concentration. The data fit a model in which enzyme-spermine and enzyme-(spermine)2 complexes are formed. A pH-independent Kd ( approximately 0.1 microM) was obtained for enzyme-spermine formation, while enzyme-(spermine)2 formation was dependent on pH; Kd at pH 6.5 = 1 microM and a Kd at pH 8 = approximately 16 microM. Direct binding of spermine was demonstrated by the ability of spermine to increase the thermal stability of the enzyme. The concentration dependence for the spermine-induced increase in thermal stability fits a model in which formation of the enzyme-spermine complex is sufficient to account for the observed changes.

Regulation of N-arginine dibasic convertase activity by amines: putative role of a novel acidic domain as an amine binding site.

Peptide sequence analysis and cDNA cloning indicate that a previously described mouse arginine-specific dibasic cleaving enzyme (dynorphin converting enzyme) [Csuhai et al. (1995) Biochemistry 34, 12411] is the homologue of N-arginine dibasic convertase (NRDc) isolated from rat testis [Chesneau et al. (1994) J. Biol. Chem. 269, 2056]. A mouse NRDc cDNA exhibited 98% amino acid identity with the rat cDNA. However, within a 74 residue acidic stretch, this identity drops to 82%. Likewise, the corresponding acidic stretch of human NRDc is only 73% identical with that of rat NRDc. To reconcile previously observed kinetic differences between rat and mouse NRDc, the hydrolysis of peptide substrates by the rat, human, and mouse enzymes was compared using phosphate and Tris as buffers. Although the three NRDc's behaved similarly, Tris had a pronounced effect on the kinetics of peptide hydrolysis. With BAM-8, alpha-neoendorphin, and dynorphin B as substrates, Tris increased KM up to 40-fold with little change in Vmax, while with dynorphin A or somatostatin 28 as substrate, Tris caused a decrease in KM of up to 100 fold, again with only a modest change in Vmax. Other amines, including the polyamines putrescine, spermidine, and spermine, all affected NRD convertase activity. It is proposed that amines bind to the acidic stretch found in NRDc, and that quantitative differences in the sensitivity to amines between the rat, mouse, and human enzymes can be at least partially accounted for by differences in their acidic stretch. The role of polyamines as physiological modulators of N-arginine dibasic convertase is considered.

Purification and characterization of a secreted arginine-specific dibasic cleaving enzyme from EL-4 cells.

A secreted dibasic cleaving peptidase capable of converting dynorphins into Leu-enkephalin-Arg6 was purified from the medium of EL-4 mouse thymoma cells. The enzyme is a novel metalloendopeptidase with a neutral pH optimum (6.9) and a molecular weight of approximately 130 000. The dibasic cleaving enzyme was completely inhibited in the presence of 20-50 mM amine buffers, 0.1 mM EDTA, 0.5 mM 1,10-phenanthroline, 0.5 mM N-ethylmaleimide, and 1mM DTNB. Unlike the Kex2 family of proteases, Ca2+ did not activate the endopeptidase, but high concentrations (1 mM) of metal ions such as Cu2+, Ni2+, Zn2+, and Co2+ completely inhibited the enzyme. Inhibition was not seen with 0.2 mM TLCK, 1 mM DTT, and 1 mM PMSF. The enzyme will cleave Arg-Arg and Arg-Lys bonds, but not Lys-Arg or Lys-Lys bonds in identical environments, and no aminopeptidase or carboxypeptidase activity was seen. The size of the substrate does not seem to be a determining factor, since dynorphin A(1-12) is cleaved at a rate similar to prodynorphin B(228-256) containing 29 amino acids. The identity of the residues on either side of the cleavage site influences the rate of processing, as noted by different rates of cleavage for the same size peptides dynorphin A(1-13) vs dynorphin A(1-9) vs beta-neoendorphin. The presence of proline in the P3' (alpha-neoendorphin), P4' (dynorphin A(1-11)), or P5' (bovine adrenal medulla dodecapeptide) position does not prevent cleavage, but neurotensin and its (1-11) fragment containing both P2 and P2' proline residues are not cleaved.

Overexpression and purification of the soluble polyhydroxyalkanoate synthase from Alcaligenes eutrophus: evidence for a required posttranslational modification for catalytic activity.

Polyhydroxyalkanoate (PHA) synthase has been expressed in Escherichia coli by reengineering the 5'-end of the wild-type (wt) gene and subsequent transformation of this gene into protease-deficient E. coli UT5600 (ompT-). Induction with IPTG results in soluble PHA synthase, which is approximately 5% of the total protein. The soluble synthase has been purified to > 90% homogeneity using FPLC chromatography on hydroxylapatite and Q-Sepharose and has a specific activity of 5 mumol min-1 mg-1. The molecular weight of the PHA product is approximately 10(6) Da based on PlGel chromatography and calibration using polystyrene molecular weight markers. The synthase in the absence of substrate appears to exist in both monomeric and dimeric forms. Incubation of the synthase with an excess of substrate converts it into a form that is now extractable into CHCl3 and sediments on sucrose density ultracentrifugation with PHA. Studies in which the ratio of substrate, 3-D-hydroxybutyrylCoA, to synthase is varied suggest that during polymerization the elongation process occurs at a rate much faster than during the initiation process. A mechanistic model has been proposed for the polymerization process [Griebel, R., Smith, Z., & Merrick, J. (1968) Biochemistry 7, 3676-3681] in which two cysteines are required for catalysis. This model is based on the well-characterized enzymes involved in fatty acid biosynthesis. To test this model, several site-directed mutants of synthase, selected based on sequence conservation among synthases, have been prepared. The C459S mutant has activity approximately 90% that of the wt protein, while the C319S and C319A synthases possess < 0.01% the activity of the wt protein. CD and antibody studies suggest that the mutant proteins are properly folded. The detection of only a single essential cysteine by mutagenesis and the requirement for posttranslational modification by phosphopantetheine to provide a second thiol in many enzymes utilizing coenzyme A thiol ester substrates made us consider the possibility that posttranslational modification was required for synthase activity as well. This hypothesis was confirmed when the plasmid containing PHA synthase (pKAS4) was transformed into E. coli SJ16, requiring beta-alanine for growth. Growth of SJ16/pKAS4 on [3H]-beta-alanine followed by Coomassie staining of the protein and autoradiography revealed that PHA synthase is overexpressed and that beta-alanine is incorporated into the protein. These results suggest PHA synthase is posttranslationally modified by phosphopantetheine.(ABSTRACT TRUNCATED AT 400 WORDS)

Mechanism of the selective functionalization of saturated hydrocarbons by Gif systems: relationship with methane monooxygenase.

Two intermediates, A and B, have been identified in the selective oxidation of saturated hydrocarbons to ketones by Gif-type systems. Intermediate A has been characterized as an Fev species with a secondary iron sigma-bond to carbon; it is captured by four different reagents or transformed into the second intermediate, B, which hydrolyzes to form a secondary alcohol. A mu-oxo Fe2III dimer is proposed as a basis for Gif-type reactivity. If the first iron is involved in the synthesis of intermediate A, the second is used to oxidize intermediate B intramolecularly to a ketal, which on hydrolysis yields a ketone. The enzyme methane monooxygenase shows a remarkable similarity to Gif-type systems in its selective hydrocarbon oxidation, particularly in the case of adamantane.