Phosphoramidon-sensitive endothelin-converting enzyme in the cytosol of cultured bovine endothelial cells

Neutral metalloproteases with endothelin-1 (ET-1) converting activity were detected in membranous and cytosolic fractions of cultured endothelial cells (EC) from bovine carotid artery in a ratio of 5:1, respectively. The cytosolic enzyme specifically and quantitatively converts big ET-1 to ET-1 (Km = 10.7 microM), but does not convert big ET-3. Like the membranous enzyme, the cytosolic enzyme is only active at pH 6.5-7.5, and is competitively inhibited by phosphoramidon (Ki = 0.79 microM). The apparent molecular weight of the cytosolic enzyme is about 540 kD, which is 5-6 times greater than that of the membranous enzyme. These results indicate the presence of two types of phosphoramidon-sensitive neutral ET-converting enzyme in vascular EC.     

Biochemical properties of endothelin converting enzyme in renal epithelial cell lines.

This is the first report clearly demonstrating the presence of endothelin (ET) converting enzyme (ECE) in non-vascular cells (renal epithelial cell lines, MDCK and LLC-PK1). ECEs derived from these epithelial cells were very similar to the endothelial ECE in the following biochemical properties: 1) The optimum pH was 7.0; 2) the Km value for big ET-1 was approximately 30 microM; 3) the enzyme was potently inhibited by EDTA, o-phenanthroline and phosphoramidon; and 4) the enzyme did not convert big ET-2 or big ET-3. These data suggest that phosphoramidon-sensitive ECE is involved in the processing of big ET-1 to ET-1 in the renal tubule.     

N-ethylmaleimide differentiates endothelin converting activity by two types of metalloproteinases derived from vascular endothelial cells

We have recently found that cultured vascular endothelial cells (ECs) contain two types of metalloproteinases which convert big endothelin-1 (big ET-1) to endothelin-1 (ET-1) via a single cleavage between Trp21 and Val22. In the present study, two enzymes were clearly differentiated by using sulfhydryl blocking reagents and anion-exchange HPLC. As reported, the converting activity of the membrane fraction of ECs was specifically inhibited by phosphoramidon. N-ethylmaleimide (NEM) markedly enhanced the apparent converting activity of the membrane fraction. This enhancement was not due to the direct action on the converting enzyme, but rather to inhibition of the degradation of big ET-1 and/or ET-1. In contrast, the converting activity of the cytosolic fraction was abolished by NEM treatment. Effects of phosphoramidon and NEM on converting activities of both fractions were confirmed after anion-exchange HPLC of each fraction, using a COSMOGEL QA column. Our results provide new information on two types of metalloproteinases which convert big ET-1 to ET-1, in vascular ECs.     

125I-endothelin-1 and 125I-big endothelin-1 in rat tissues: autoradiographic localization and receptor binding.

The potent vasoconstrictor peptide, endothelin-1 (ET-1), which exhibits a characteristically long-acting activity in vitro and in vivo, is thought to be generated in endothelial cells from a less active intermediate, big endothelin-1 (big ET-1). In addition to ET-1, big ET-1 is also present in the circulation. The autoradiographic localization of 125I-big ET-1 and 125I-ET-1 has been studied after intravenous administration in rat tissues. Highest enrichment of radioactivity was found in the kidney cortex for both peptides. Compared to blood levels, enrichment of radioactivity is also detected, in the vascular wall of the aorta. Comparing the radioactivity pattern of ET-1 and big ET-1, a nearly identical tissue distribution is observed, with the exception of the relative enrichment in the lung and the zona glomerulosa after administration of ET-1. Both radioligands show a specific and saturable binding to lung and kidney membranes. In the case of lung tissue, Ki values are 10(-10) M for endothelin-1 and 10(-8) M for big endothelin-1. This difference in affinities may account for the lack of binding of big endothelin-1 to lung tissue.     

Phosphoramidon inhibits the conversion of intracisternally administered big endothelin-1 to endothelin-1

It is suggested that endothelin-1 (ET-1), a potent vasoconstrictor peptide, is involved in the pathogenesis of cerebral vasospasm following subarachnoid hemorrhage (SAH). We examined the effects of intracisternal administration of big ET-1 on the cerebral arteries in the absence or presence of pretreatment with phosphoramidon, an inhibitor of ET converting enzyme, in anesthetized dogs. After intracisternal administration of big ET-1 (10 micrograms/dog), the caliber of the basilar artery on the angiogram was decreased to about 59% of the control. This was accompanied by a marked increase in immunoreactive ET in the cerebrospinal fluid. Systemic arterial pressure was markedly elevated following big ET-1 injection. All changes induced by big ET-1 were effectively prevented with phosphoramidon. These data suggest that intracisternally administered big ET-1 is converted to ET-1 and that the generated ET-1 produces cerebral vasospasm and hypertension. A phosphoramidon-sensitive metalloproteinase appears to contribute to this conversion.     

Do the structures of big ET-1 and big ET-3 adopt a similar overall fold? Consequences for endothelin converting enzyme specificity

Big ET-1 and big ET-3 are precursor peptides which render endothelin-1 (ET-1) and endothelin-3 (ET-3) relatively unreactive and resistant to proteolytic cleavage. Big ET-1 is cleaved in vivo by ECE-1 (endothelin-converting enzyme), and big ET-3 is also cleaved but apparently to a significantly lesser extent by this enzyme. To shed light on the relation between structure and function, circular dichroism (CD) spectroscopy and homology modeling were used to determine whether big ET-1 and big ET-3 adopt similar secondary and tertiary structures. Analyses of the CD spectra and thermal denaturation indicate they have similar secondary structures and thermal stabilities. Superposition of the modeled coordinates of both peptides indicates that they can adopt the same overall fold except in the C-terminal residues, 34-38 in big ET-1 and 34-41 in big ET-3. This region corresponds to an area of complete sequence heterogeneity between the two peptides. A model has been developed which has a loop for residues 27-30 (HVVP in big ET-1), which have previously been demonstrated to be essential for eliciting efficient hydrolysis of the W21-V22 bond in big ET-1 and which have the sequence QTVP in big ET-3. Differences in affinity between big ET-1 and big ET-3 for ECE-1 thus appear to be due solely to sequence variations in the local region of the cleavage site.     

Identification and partial purification of a thiol endothelin-converting enzyme from porcine aortic endothelial cells.

Endothelin is a potent peptide vasoconstrictor. The final step in the processing of endothelin has been postulated to be the cleavage of the Trp21-Val22 peptide bond in proendothelin by a putative endothelin-converting enzyme. A soluble extract of primary porcine aortic endothelial cells was found to contain an enzyme activity that converted proendothelin-1 (proET-1) to an endothelin-1 (ET-1)-like peptide as determined by the rabbit aortic ring contraction assay. This enzyme was partially purified by DE52 ion-exchange chromatography. Incubation of proET-1 with the partially purified enzyme generated a product which had a retention time on HPLC identical to that of authentic ET-1. Further analysis of the product showed that it caused contraction of rabbit aortic rings, had a molecular weight identical to ET-1 as measured by fast atom bombardment mass spectrometry, and competed for [125I]ET-1 binding in an RIA using specific antibodies which recognize the carboxy terminal tryptophan of ET-1. The enzyme activity could be inhibited by thiol protease inhibitors such as Z-phe-pheCHN2 and p-hydroxymercuribenzoate, but not by serine- or metalloprotease inhibitors. The optimal pH for the enzymatic activity was between 7.0 and 7.5, and no activity was detected at pH 4.0. These results demonstrate that this thiol protease is a potential endothelin-converting enzyme.     

Kinetic parameters of the protein tyrosine kinase activity of EGF-receptor mutants with individually altered autophosphorylation sites.

Epidermal growth factor (EGF)-receptor mutants in which individual autophosphorylation sites (Tyr1068, Tyr1148 or Tyr1173) have been replaced by phenylalanine residues were expressed in NIH-3T3 cells lacking endogenous EGF-receptors. Kinetic parameters of the kinase of wild-type and mutant receptors were compared. Both wild-type and mutant EGF-receptors had a Km(ATP) 1-3 microM for the autophosphorylation reaction, and a Km(ATP) of 3-7 microM for the phosphorylation of a peptide substrate. These are similar to the Km(ATP) values reported for EGF-receptor of A431 cells. A synthetic peptide representing the major in vitro autophosphorylation site Tyr1173 of the EGF-receptor (KGSTAENAEYLRV) was phosphorylated by wild-type receptor with a Km of 110-130 microM, and the peptide inhibited autophosphorylation with a Ki of 150 microM. Mutant EGF-receptors phosphorylated the peptide substrate with a Km of 70-100 microM. A similar decrease of Km (substrate) was obtained when the phosphorylation experiments were performed with the commonly applied substrates angiotensin II and a peptide derived from c-src. The Km of angiotensin II phosphorylation was reduced from 1100 microM for wild-type receptor to 890 microM for mutant receptor and for c-src peptide from 1010 microM to 770 microM respectively. The Vmax of the kinase was dependent on receptor concentration, but was not significantly affected by the mutation. Analogs of the Tyr1173 peptide in which the tyrosine residue was replaced by either a phenylalanine or an alanine residue also inhibited autophosphorylation with Ki of 650-750 microM. These analyses show that alterations of individual autophosphorylation sites do not have a major effect on kinase activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

125I-endothelin-1 and 125I-big endothelin-1 in rat tissues: autoradiographic localization and receptor binding

Summary The potent vasoconstrictor peptide, endothelin-1 (ET-1), which exhibits a characteristically long-acting activity in vitro and in vivo, is thought to be generated in endothelial cells from a less active intermediate, big endothelin-1 (big ET-1). In addition to ET-1, big ET-1 is also present in the circulation. The autoradiographic localization of ¹²⁵I-big ET-1 and ¹²⁵I-ET-1 has been studied after intravenous administration in rat tissues. Highest enrichment of radioactivity was found in the kidney cortex for both peptides. Compared to blood levels, enrichment of radioactivity is also detected, in the vascular wall of the aorta. Comparing the radioactivity pattern of ET-1 and big ET-1, a nearly identical tissue distribution is observed, with the exception of the relative enrichment in the lung and the zona glomerulosa after administration of ET-1.Both radioligands show a specific and saturable binding to lung and kidney membranes. In the case of lung tissue, K _i values are 10^–10 M for endothelin-1 and 10^–8 M for big endothelin-1. This difference in affinities may account for the lack of binding of big endothelin-1 to lung tissue.     

Phosphoramidon-sensitive endothelin-converting enzyme in vascular endothelial cells converts big endothelin-1 and big endothelin-3 to their mature form.

Incubation of big endothelin-3 (big ET-3(1-41)) with the membrane fraction obtained from cultured endothelial cells (ECs) resulted in an increase in immunoreactive-ET (IR-ET). This increasing activity was markedly suppressed by phosphoramidon, which is known to inhibit the conversion of big ET-1(1-39) to ET-1(1-21). Reverse-phase HPLC of the incubation mixture of the membrane fraction with big ET-3 revealed one major IR-ET component corresponding to the elution position of synthetic ET-3(1-21). When the cultured ECs were incubated with big ET-3, a conversion to the mature ET-3, as well as an endogenous ET-1 generation, was observed. Both responses were markedly suppressed by phosphoramidon. By the gel filtration of 0.5% CHAPS-solubilized fraction of membrane pellets of ECs, the molecular mass of the proteinase which converts big ET-1 and big ET-3 to their mature form was estimated to be 300-350 kDa. Phosphoramidon almost completely abolished both converting activities of the proteinase. We conclude that the above type of phosphoramidon-sensitive metalloproteinase functions as an ET-converting enzyme to generate the mature form from big ET-1 and big ET-3 in ECs.     

The biosynthesis of endothelin-1 by human polymorphonuclear leukocytes

Human polymorphonuclear leukocytes (PMNs) converted human big endothelin (bET; 2 microM) to an endothelin-1 (ET-1) like contractile factor, as assessed by bioassay. The generation of this ET-1 like activity was rapid (minutes), time-dependent and more pronounced in non-activated cells, suggesting a partial degradation by activated PMNs. Phosphoramidon (54 micrograms/ml) inhibited the formation of this contractile factor, whereas phenylmethylsulfonylfluoride (PMSF; 25 micrograms/ml), pepstatin A (1 microgram/ml) or epoxysuccinyl-L-leucylamido-(guanidino)butane (E-64; 10 micrograms/ml) did not. Incubations of activated PMNs with PMSF significantly potentiated the generation of ET-1 like activity and selectively inhibited the degradation of [125I]ET-1 by activated PMNs. These findings indicate that human PMNs contain and/or release neutral proteases, which can both rapidly produce and degrade ET-1, an observation which may have important (patho)physiologic implications.     

Cyclic expression of endothelin-converting enzyme-1 mediates the functional regulation of seminiferous tubule contraction.

The potent smooth muscle agonist endothelin-1 (ET-1) is involved in the local control of seminiferous tubule contractility, which results in the forward propulsion of tubular fluid and spermatozoa, through its action on peritubular myoid cells. ET-1, known to be produced in the seminiferous epithelium by Sertoli cells, is derived from the inactive intermediate big endothelin-1 (big ET-1) through a specific cleavage operated by the endothelin-converting enzyme (ECE), a membrane-bound metalloprotease with ectoenzymatic activity. The data presented suggest that the timing of seminiferous tubule contractility is controlled locally by the cyclic interplay between different cell types. We have studied the expression of ECE by Sertoli cells and used myoid cell cultures and seminiferous tubule explants to monitor the biological activity of the enzymatic reaction product. Northern blot analysis showed that ECE-1 (and not ECE-2) is specifically expressed in Sertoli cells; competitive enzyme immunoassay of ET production showed that Sertoli cell monolayers are capable of cleaving big ET-1, an activity inhibited by the ECE inhibitor phosphoramidon. Microfluorimetric analysis of intracellular calcium mobilization in single cells showed that myoid cells do not respond to big endothelin, nor to Sertoli cell plain medium, but to the medium conditioned by Sertoli cells in the presence of big ET-1, resulting in cell contraction and desensitization to further ET-1 stimulation; in situ hybridization analysis shows regional differences in ECE expression, suggesting that pulsatile production of endothelin by Sertoli cells (at specific "stages" of the seminiferous epithelium) may regulate the cyclicity of tubular contraction; when viewed in a scanning electron microscope, segments of seminiferous tubules containing the specific stages characterized by high expression of ECE were observed to contract in response to big ET-1, whereas stages with low ECE expression remained virtually unaffected. These data indicate that endothelin-mediated spatiotemporal control of rhythmic tubular contractility might be operated by Sertoli cells through the cyclic expression of ECE-1, which is, in turn, dependent upon the timing of spermatogenesis.     

Influence of the lactokinin Ala-Leu-Pro-Met-His-Ile-Arg (ALPMHIR) on the release of endothelin-1 by endothelial cells

Milk protein-derived peptides with angiotensin-converting enzyme (ACE) inhibitory activity can reduce blood pressure in hypertensive subjects. The lactokinin Ala-Leu-Pro-Met-His-Ile-Arg (ALPMHIR) is an ACE-inhibitory peptide released by tryptic digestion from the milk protein beta-lactoglobulin. Its ACE-inhibitory activity is 100 times lower than that of captopril. The latter is known to inhibit the release of the vasoconstrictor endothelin-1 (ET-1) by endothelial cells. The effects of ALPMHIR on the endothelium are currently unknown. In this study, the influence of ALPMHIR on release of ET-1 by endothelial cells was investigated. The basal ET-1 release of the cells was reduced by 29% (p<0.01) in the presence of 1 mM ALPMHIR, compared to 42% (p<0.01) for 0.1 mM captopril. Addition of 10 U/ml thrombin to the incubation medium increased the release of ET-1 by 66% (p<0.01). Co-incubation of 10 U/ml thrombin with 1 microM captopril or with 0.1 mM ALPMHIR inhibited the stimulated ET-1 release by 45% (p<0.01) and by 32% (p<0.01), respectively. These data indicate that dietary peptides, such as ALPMHIR, can modulate ET-1 release by endothelial cells. These effects, among other mechanisms, may play a role in the anti-hypertensive effect of milk protein-derived peptides.     

Cultured bovine endothelial cells convert big endothelin isopeptides to mature endothelin isopeptides

The incubation of big endothelin-1 (big ET-1), big ET-2 or big ET-3 with cultured bovine endothelial cells (ECs) resulted in their conversions to mature endothelins (ETs). These conversions apparently exhibited Michaelis-Menten kinetics as a function of each big ET isopeptide. The conversions of big ETs were abolished by phosphoramidon. These results indicate that vascular endothelium can convert exogenous big ET-1 to mature ET-1 through a phosphoramidon-sensitive metalloprotease, and that this enzyme has also high affinities for big ET-2 and big ET-3.     

A membrane-bound metallo-endopeptidase from rat kidney. Characteristics of its hydrolysis of peptide hormones and neuropeptides.

A membrane-bound metallo-endopeptidase that hydrolyzes human parathyroid hormone (1-84) and reduced hen egg lysozyme between hydrophilic amino acid residues was isolated from rat kidney [Yamaguchi et al. (1991) Eur. J. Biochem. 200, 563-571]. In this study, the hydrolyses of various peptide hormones and neuropeptides by the metallo-endopeptidase were examined using an automated gas-phase protein sequencer. The purified enzyme hydrolyzed the oxidized insulin B chain and substance P most rapidly, followed by big endothelin 1, neurotensin, angiotensin 1, endothelin 1, rat alpha-atrial natriuretic peptide and bradykinin, in this order. The enzyme mainly cleaved these peptides at bonds involving a hydrophilic amino acid residue. However, it cleaved bonds between less hydrophilic amino acid pairs in several short peptides, e.g. at the His5-Leu6 bond in oxidized insulin B chain, the Ile28-Val29 bond in big endothelin-1 and the Ile5-His6 and Phe8-His9 bonds in angiotensin 1. The enzyme cleavage sites of oxidized insulin B chain and angiotensin 1 were different from the reported sites cleaved by meprin and by endopeptidase 2, respectively. Kinetic determination of bradykinin hydrolysis by the purified enzyme yielded values of Km = 18.1 microM and kcat = 0.473 s-1, giving a ratio of kcat/Km = 2.62 x 10(4) s-1.M-1. The Km value was about 20-fold lower than that reported for meprin and endopeptidase 2. These results indicate that the membrane-bound metallo-endopeptidase from rat kidney is distinguished from meprin and endopeptidase 2 in its substrate specificity and is not parathyroid hormone specific, but has potential capacities to inactivate various biologically active peptide hormones and neuropeptides in vivo.     

Characterization of SARS main protease and inhibitor assay using a fluorogenic substrate

SARS main protease is essential for life cycle of SARS coronavirus and may be a key target for developing anti-SARS drugs. Recently, the enzyme expressed in Escherichia coli was characterized using a HPLC assay to monitor the formation of products from 11 peptide substrates covering the cleavage sites found in the SARS viral genome. This protease easily dissociated into inactive monomer and the deduced Kd of the dimer was 100 microM. In order to detect enzyme activity, the assay needed to be performed at micromolar enzyme concentration. This makes finding the tight inhibitor (nanomolar range IC50) impossible. In this study, we prepared a peptide with fluorescence quenching pair (Dabcyl and Edans) at both ends of a peptide substrate and used this fluorogenic peptide substrate to characterize SARS main protease and screen inhibitors. The fluorogenic peptide gave extremely sensitive signal upon cleavage catalyzed by the protease. Using this substrate, the protease exhibits a significantly higher activity (kcat = 1.9 s(-1) and Km = 17 microM) compared to the previously reported parameters. Under our assay condition, the enzyme stays as an active dimer without dissociating into monomer and reveals a small Kd value (15 nM). This enzyme in conjunction with fluorogenic peptide substrate provides us a suitable tool for identifying potent inhibitors of SARS protease.     

Disulfide bonds in big ET-1 are essential for the specific cleavage at the Trp(21)-Val(22) bond by soluble endothelin converting enzyme-1 from baculovirus/insect cells

Endothelin converting enzyme-1 (ECE-1) is a type II integral membrane protein and a zinc metalloendopeptidase. ECE-1 generates endothelin-1 (ET-1), the most potent vasoconstrictor yet discovered, by specific proteolytic processing of a precursor peptide, big ET-1. An insect cell expression system, which generates up to 4.3 mg of a secreted, soluble form of ECE-1 (solECE-1) per liter culture medium, has been established and solECE-1 was purified to homogeneity using five chromatographic steps. SolECE-1 expressed in insect cells could be suitable for X-ray structure determination as it is much less glycosylated than solECE-1 from mammalian cells. SolECE-1 from both sources, nonetheless, has comparable enzymatic properties. Despite apparent structural similarities, ECE-1 cleaves big ET-1 exclusively between Trp(21) and Val(22), in contrast to neprilysin, which cleaves big ET-1 at various sites. However, when linear big ET-1, in which the formation of disulfide bonds has been prevented by alkylation of the four cysteines, was used as substrate, it was cleaved by solECE-1 at multiple sites. This result indicates that secondary/tertiary structure of big ET-1 induced by disulfide bonds is essential for the specific cleavage of the Trp(21)-Val(22) bond by ECE-1. A continuous, fluorescent ECE-1 assay has been developed using a novel substrate, 2-aminobenzoyl-Arg-Pro-Pro-Gly-Phe-Ser-Pro-(p-nitro-Phe(8))-Arg. This simple and rapid assay can greatly facilitate discovery of novel ECE inhibitors useful as pharmaceutical agents.     

Tyrosine protein kinase activity in the DMBA-induced rat mammary tumor: inhibition by quercetin

Tyrosine protein kinase activity was measured in membranes from DMBA-induced mammary tumors, with Angiotensin II as substrate. The apparent Km for the peptide was 3.3 mM. This enzymatic activity is inhibited by Ca+2; Mn+2 can replace Mg+2 with an increase in the Km for ATP from 47 /microM to 172 microM. The enzymatic activity was not affected by cyclic AMP but was inhibited in dose dependent manner by quercetin, a bioflavonoid which is known to inhibit proliferation of malignant cells in vitro.     

Proliferative effects of angiotensin II and endothelin-1 on guinea pig gingival fibroblast cells in culture

We investigated whether phenytoin (PHT) and nifedipine (NIF) induce angiotensin II (Ang II) and endothelin-1 (ET-1) generation by cultured gingival fibroblasts derived from guinea pigs and whether Ang II and ET-1 induce proliferation of these cells. Immunohistochemical experiments showed that PHT (250 nM) and NIF (250 nM) increased the immunostaining intensities of immunoreactive Ang II and ET-1 (IRET-1) in these cells. Captopril (3 microM), an angiotensin-converting enzyme inhibitor, reduced these enhanced intensities to control levels. Ang II (100 nM) enhanced the immunostaining intensity of IRET-1. PHT (250 nM) and NIF (250 nM)-induced cell proliferation. Both PHT- and NIF-induced proliferation was inhibited by captopril (3 microM). Ang II (100 nM) and ET-1 (100 nM) also induced cell proliferation. Ang II-induced proliferation was inhibited by CV11974 (1 microM), an AT(1) receptor antagonist and saralasin (1 microM), an AT(1)/AT(2) receptor antagonist, but not by PD123,319 (1 microM), an AT(2) receptor antagonist. ET-1-induced proliferation was inhibited by BQ123 (10 microM), an ET(A) receptor antagonist, but not by BQ788 (1 microM), an ET(B) receptor antagonist. These findings suggest that PHT- and NIF-induced gingival fibroblast proliferation is mediated indirectly through the induction of Ang II and ET-1 and probably mediated through AT(1) and ET(A) receptors present in or on gingival fibroblasts.     

Processive action of the two peptide binding sites of prolyl 4-hydroxylase in the hydroxylation of procollagen

The number of peptide binding sites of prolyl 4-hydroxylase was manipulated with the peptide photoaffinity label N-(4-azido-2-nitrophenyl)glycyl-(Pro-Pro-Gly)5, and the effect on hydroxylation of the relatively short peptide substrate (Pro-Pro-Gly)5 and of the long natural substrate procollagen was studied. With (Pro-Pro-Gly)5 as a substrate, a linear relation was found between enzyme activity and the amount of covalently bound photoaffinity label, approximately 50% inactivation being reached at 1 mol of label/mol of enzyme. No difference in Km value for (Pro-Pro-Gly)5 was detected between unlabeled and partially labeled enzyme preparations. These results indicate that enzyme molecules with only one free active site hydroxylated the synthetic substrate (Pro-Pro-Gly)5 with the same Km and at half the rate of native enzyme. In contrast, with procollagen as a substrate a 5-10-fold increase in Km was found with the fraction of enzyme containing only one free active site, as compared to the Km for procollagen with nonlabeled enzyme. This finding is explained by an enzyme-kinetic model based on a processive action of the two peptide substrate binding sites of prolyl 4-hydroxylase, preventing dissociation of the enzyme-substrate complex between successive hydroxylations of a long peptide with multiple substrate sites. Such a mechanism leads to a low Km for a long peptide by overcoming the diffusional constraints on the rate of association between the enzyme and the individual substrate sites.