Biological effects of human gastrin I and II chemically modified at the C-terminal tetrapeptide amide.

Binding to gastrin receptors and gastric acid secretion experiments were performed with gastrin derivatives modified at the C-terminal tetrapeptide amide from HG-13 sequence. 1. When the ultimate phenylalanine amide was replaced by a phenethylester or a phenetylamide moiety, the resulting compound bound to gastrin receptors (Kd approximately 10 nM) and exhibited antagonist activity on gastrin-induced acid secretion in the anesthetized rat. 2. Changing the peptide bond between Trp and Leu residues to a -omega(CH2-NH)- bond resulted in a compound which also bound to gastrin receptors (Kd approximately 10 nM) but presented agonist activity on acid secretion in the rat. In contrast, when the peptide bond between Leu and Asp residues was replaced by a -omega(CH2-NH)- bond, the resulting compound was devoid of any affinity for gastrin receptor (Kd greater than 10(-6) M) and of any biological activity. 3. The HG-13 derivatives were synthesized in sulfated and unsulfated forms: O-sulfation of the HG-13 tyrosine residue did not change its intrinsic in vivo activity but enhanced its affinity for gastrin receptors (Kd approximately 0.3 nM). On the contrary, O-sulfation of the various chemically modified HG-13 had no significant effect in either in vitro or in vivo experiments. 4. Finally, no significant difference between binding on parietal (F3) and nonparietal (F1) cells was observed, in agreement with the presence of a gastrin-type receptor in these two cell populations.     

Phenylethylamide derivatives of the C-terminal tetrapeptide of gastrin. Potent inhibitors of gastrin-stimulated acid secretion

Peptide analogues of the C-terminal tetrapeptide of gastrin in which the phenylalanine had been replaced were synthesized and their biological activity on acid secretion evaluated. Compounds Boc-Trp-Leu-Asp phenylethylamide 6, Boc-beta-Ala-Trp-Leu-Asp phenylethylamide 9, Boc-Trp-Leu-Asp p-fluorophenylethylamide 19, Boc-Trp-psi(CH2NH)-Leu-Asp phenylethylamide 23, Boc-Trp-Leu-Asp 2,2-diphenylethylamide 15, and Boc-D Trp-Leu-Asp 2,2-diphenylethylamide 21, in which the phenylalanine had been replaced by phenylethylamine, p-fluorophenylethylamine or 2,2-diphenylethylamine were synthesized. None of these derivatives showed activity on acid secretion in the anaesthetized rat at doses as high as 5 mg/kg. However, they were potent inhibitors of gastrin-induced acid secretion, with ED50 varying from 0.1 to 0.6 mg/kg.     

Potent trypsin-resistant hGH-RH analogues.

A series of analogues of hGH-RH-(1-29)-NH2 designed to have metabolic stability has been synthesized. Standard Boc-SPPS was employed, modified to permit the guanidinylation of amino side-chains after chain assembly but before release from the resin. [Dat1, Har(11, 12, 20, 21, 29), Ala15, Nle27, Asp28]-, [Dat1, Har(11, 20, 29), Orn12, Ala15, Nle27, Asp28]-, and [Dat1, Gap(11,12, 21, 29), Ala15, Har20, Nle27, Asp28]-hGH-RH-(1-29)-NH2 were completely resistant to trypsin and about 50 times as potent as hGH-RH-(1-29)-NH2 itself when injected subcutaneously in rats. These peptides are candidates for clinical application in the therapy of GH deficiency.     

Methoxinine – an alternative stable amino acid substitute for oxidation‐sensitive methionine in radiolabelled peptide conjugates

Radiolabelled peptides with high specificity and affinity towards receptors that are overexpressed by tumour cells are used in nuclear medicine for the diagnosis (imaging) and therapy of cancer. In some cases, the sequences of peptides under investigations contain methionine (Met), an amino acid prone to oxidation during radiolabelling procedures. The formation of oxidative side products can affect the purity of the final radiopharmaceutical product and/or impair its specificity and affinity towards the corresponding receptor. The replacement of Met with oxidation resistant amino acid analogues, for example, norleucine (Nle), can provide a solution. While this approach has been applied successfully to different radiolabelled peptides, a Met → Nle switch only preserves the length of the amino acid side chain important for hydrophobic interactions but not its hydrogen‐bonding properties. We report here the use of methoxinine (Mox), a non‐canonical amino acid that resembles more closely the electronic properties of Met in comparison to Nle. Specifically, we replaced Met¹⁵ by Mox¹⁵ and Nle¹⁵ in the binding sequence of a radiometal‐labelled human gastrin derivative [d ‐Glu¹⁰]HG(10‐17), named MG11 (d ‐Glu‐Ala‐Tyr‐Gly‐Trp‐Met‐Asp‐Phe‐NH₂). A comparison of the physicochemical properties of ¹⁷⁷Lu‐DOTA[ X ¹⁵]MG11 ( X = Met, Nle, Mox) in vitro (cell internalization/externalization properties, receptor affinity (IC₅₀), blood plasma stability and logD) showed that Mox indeed represents a suitable, oxidation‐stable amino acid substitute of Met in radiolabelled peptide conjugates. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

External pancreatic secretion in the rat: supramaximal inhibition induced by the cholecystokinin octapeptide [CCK(26-33)] and analogs altered on the 28-29 bond

Supramaximal doses of cholecystokinin induce in vitro submaximal biological responses, desensitization and residual stimulation. In vivo, supramaximal inhibition and oedematous pancreatitis have been reported. The aim of this study was to analyze the in vivo response of the pancreatic secretion of the rat to a wide range of doses of CCK8 and analogs prepared by alterations of the Met(28)-Gly(29) bond, a modification that may lead to potent agonists. We used Boc-[Nle28-Nle31]-CCK(26-33) (1) and derivatives of (1) with the 28-29 peptide bond replaced by CH2-NH (2), CO-CH2 (3), CH2-CH2 (4), NH-CO (5). On infusions, the ED50's (pmol/kg.min) for protein output were 4 for CCK8 and (1), 11 for (3), 40 for (2) and (4), and 860 for (5). The relative order of the in vivo potencies was near to the one determined in vitro on isolated rat acini. On bolus injections, the maximal response was observed with 300 pmol/kg of CCK8, and peaked 10-15 min after the injection. With higher doses of CCK8, the secretory peak was smaller, and was delayed relative to the moment of the injection. Supramaximal doses of CCK analogs induced the same pattern of response; however, the peak injection delay was in some cases smaller than after CCK8. Determination of the plasma CCK levels indicated that the time of peak effect after supramaximal doses of CCK8 was delayed relative to the time of effective maximal plasma CCK levels. This suggests a slow dissociation of CCK8 from one of its pancreatic binding sites in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis of analogues of the Des-Phe-NH2 C-terminal hexapeptide of cholecystokinin showing gastrin antagonist activity

Four analogues of Z-CCK-27-32-NH2, Z-Tyr(SO3-)-Met-Gly-Trp-Met-Asp-NH2, a cholecystokinin receptor antagonist have been synthesized by solution methodology. In these analogues, Z-Tyr(SO3-)-Nle-Gly-Trp-Met-Asp-NH2 16, Z-Tyr(SO3-)-Nle-Gly-Trp-Nle-Asp-NH2 17, BOC-Tyr(SO3-)-Met-Gly-Trp-Met-Asp-NH2 24 and Boc-Tyr(SO3-)-Met-Gly-Trp-Nle-Asp-NH2 25 methionyl residues were replaced by norleucyl residues. Preliminary biological activity on gastrin-induced acid secretion, in rat, are reported. These derivatives proved to antagonize the action of gastrin, with ED 50 of between 0.5 and 3 mg/kg.     

Multiple cleavage sites of cholecystokinin heptapeptide by "enkephalinase"

Degradation of Boc CCK7 (Boc Tyr1 (SO3H)-Met2-Gly3-Trp4-Met5-Asp6-Phe7-NH2), a fully active analog of CCK8, by purified rabbit kidney neutral metalloendopeptidase (enkephalinase) was studied as a basis for the rational design of potent peptidases-resistant analogs of cholecystokinin. Characterization of the metabolites was performed by HPLC using several elution procedures. Three cleavage sites were evidenced: one major at the Asp6-Phe7 bond and two minor at Gly3-Trp4 and Trp4-Met5 bonds. All cleavages were fully inhibited by thiorphan, a potent inhibitor of enkephalinase. The relative importance of the different cleavages was established using several cholecystokinin analogs. At 25 degrees C the half-disappearance time was 18 min for Boc CCK7, Boc[diNle2,5]CCK7 and 70 min for Boc[diNle2,5 D.Asp6]CCK7. Although, half-life of Boc CCK7 and Boc[diNle2,5]CCK7 were identical, the replacement of Met by Nle, a more hydrophobic aminoacid, greatly favoured the cleavage at the Trp4-Nle5 bond which became the major breakdown. This feature was exemplified by the substitution of L.Asp by D.Asp, preventing the Trp4-Nle5 cleavage, which gave rise to the most enkephalinase-resistant analog in this series.     

Potent agonists of growth hormone-releasing hormone. Part I.

Analogs of the 29 amino acid sequence of growth hormone-releasing hormone (GH-RH) with agmatine (Agm) in position 29 have been synthesized by the solid phase method, purified, and tested in vitro and in vivo. The majority of the analogs contained desaminotyrosine (Dat) in position 1, but a few of them had Tyr1, or N-MeTyr1. Some peptides contained one or more additional L- or D-amino acid substitutions in positions 2, 12, 15, 21, 27, and/or 28. Compared to the natural sequence of GH-RH(1-29)NH2, [Dat1,Ala15]GH-RH(1-28)Agm (MZ-3-191) and [D-Ala2,Ala15]GH-RH(1-28)Agm (MZ-3-201) were 8.2 and 7.1 times more potent in vitro, respectively. These two peptides contained Met27. Their Nle27 analogs, [Dat1,Ala15,Nle27]GH-RH(1-28)Agm(MZ-2-51), prepared previously (9), and [D-Ala2,Ala15,Nle28]GH-RH(1-28)Agm(MZ-3-195) showed relative in vitro potencies of 10.5 and 2.4, respectively. These data indicate that replacement of Met27 by Nle27 enhanced the GH-releasing activity of the analog when the molecule contained Dat1-Ala2 residues at the N-terminus, but peptides containing Tyr1-D-Ala2 in addition to Nle27 showed decreased potencies. Replacement of Ser28 with Asp in multi-substituted analogs of GH-RH(1-28)Agm resulted in a decrease in in vitro potencies compared to the parent compound. Thus, the Ser28-containing MZ-2-51, and [Dat1,Ala15,D-Lys21,Nle27]GH-RH(1-28)Agm, its Asp28 homolog (MZ-3-149), possessed relative activities of 10.5 and 5.6, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conformational-functional relationships of tetragastrin analogs]

Sets of low-energy backbone conformations of the active tetragastrin analogue Boc-Trp-Leu-Asp-Phe-NH2 and two competitive antagonists Boc-Trp-Leu psi (CH2NH)-Asp-Phe-NH2 and Boc-Trp-Leu-Asp-O-CH2-CH2-C6H5 were obtained using theoretical conformational analysis methods. Groups of the conformations were selected for the three analogues, allowing a spatial matching of Trp, Asp and Phe residues responsible for the gastrin receptor binding. Three conformations possessing the lowest energies among the geometrically similar structures of these three peptides are suggested as a model for the "receptor-bound" conformations of these analogues. Backbone spatial folding resembling an alpha-helix turn is characteristic of these conformations. The correspondence of the proposed model to the available data on structure--activity relationships for tetragastrin analogues is discussed. Orientations of the putative receptor-bound conformations in a "water--lypophylic medium" two-phase system were investigated.     

The role of the Asp-32 residue of cholecystokinin in gastric acid secretion and gastrin receptor recognition

The aspartic acid residue at the penultimate position is known to be essential for the hormonal activity of CCK and gastrin on gastric acid secretion. This residue was successively replaced by beta-aspartic acid, beta-alanine, and glutamic acid in the C-terminal heptapeptide of CCK 27-33. The analogues obtained were tested on rat gastric acid secretion and for recognition by gastrin receptors. The replacement by beta-aspartic or beta-alanine decreased gastric secretion and gastrin receptor recognition. In contrast, replacement by glutamic acid affected these two parameters less. The nature of the N-blocking group (Boc or Z) also influenced these activities, Boc derivatives being more potent than Z derivatives. The results were compared to those previously obtained on pancreatic secretion and on stimulation of gall bladder contraction where the modifications were found capable of differentiating between cholecystokinin, pancreozymin and gastrin activities.     

Conformational and molecular modeling studies of beta-cyclodextrin-heptagastrin and the third extracellular loop of the cholecystokinin 2 receptor

The conformational features of a conjugate of the C-terminus of human gastrin (HG[11-17]), the shortest gastrin sequence retaining biological function, with beta-cyclodextrin ([Nle(15)]-HG[11-17]-betaCD) were determined by NMR spectroscopy in an aqueous solution of dodecylphosphocholine (DPC) micelles. The peptide-betaCD conjugate displays a binding affinity and activation profile comparable to those of HG[11-17] at the cholecysokinin 2 (CCK(2)) receptor, the G protein-coupled receptor responsible for the gastrointestinal function of gastrin. The structure of the peptide consisted of a well-defined beta-turn between Gly(13) and Asp(16) of gastrin. The structural preferences of [Nle(15)]-HG[11-17]-betaCD in DPC micelles and the 5-doxylstearate-induced relaxation of the (1)H NMR resonances support a membrane-associated receptor recognition mechanism. Addition of [Nle(15)]-HG[11-17]-betaCD to the third extracellular loop domain of the CCK(2) receptor, CCK(2)-R(352-379), generated a number of intermolecular nuclear Overhauser enhancements (NOEs) and chemical shift perturbations. NOE-restrained MD simulations of the [Nle(15)]-HG[11-17]-betaCD-CCK(2)-R complex produced a topological orientation in which the C-terminus was located in a shallow hydrophobic pocket near the confluence of TM2 and -3. Despite the steric bulk and physicochemical properties of betaCD, the [Nle(15)]-HG[11-17]-betaCD-CCK(2)-R complex is similar to the CCK-8-CCK(2)-R complex determined previously, providing insight into the mode of ligand binding and the role of electrostatic interactions.     

Cyclic lactam analogues of Ac-[Nle4]alpha-MSH4-11-NH2

Two side-chain cyclic lactam analogues of the 4-11 fragment of alpha-melanocyte-stimulating hormone (alpha-MSH), Ac-[Nle4,D-Orn5,Glu8]alpha-MSH4-11-NH2 and Ac-[Nle4,D-Orn5,D-Phe7,Glu8]alpha-MSH4-11-NH2, were prepared on p-methylbenzhydrylamine resin by using a combination of N alpha-Boc and N alpha-Fmoc synthetic strategies with diphenyl phosphorazidate mediated cyclization. The melanotropin activities of these two analogues were examined and compared relative to those of alpha-MSH, Ac-[Nle4]alpha-MSH4-11-NH2, and Ac-[Nle4,D-Phe7]alpha-MSH4-11-NH2. In the frog (Rana pipiens) skin bioassay, the L-Phe7 17-membered ring cyclic analogue was slightly more potent than the linear Ac-[Nle4]alpha-MSH4-11-NH2 and exhibited prolonged melanotropic bioactivity (greater than or equal to 4 h). In this same assay, the D-Phe7 cyclic analogue was more than 100-fold less potent than the L-Phe cyclic analogue and was 10,000 times less potent than linear Ac-[Nle4,D-Phe7]alpha-MSH4-11-NH2. In the lizard skin (Anolis carolinensis) bioassay, the L-Phe7 cyclic analogue was 100-fold less potent than Ac-[Nle4]alpha-MSH4-11-NH2, while the D-Phe7 cyclic analogue was 10,000-fold less potent than both Ac-[Nle4]alpha-MSH4-11-NH2 and the D-Phe7 linear derivative Ac-[Nle4,D-Phe7]alpha-MSH4-11-NH2. The solution conformation of these two cyclic analogues in dimethyl sulfoxide-d6 was examined by 1D and 2D 500-MHz 1H NMR spectroscopy. Our analysis suggests an H bond stabilized C10 (or C13) turn for the D-Phe7 cyclic structure while the L-Phe7 analogue is more conformationally flexible.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis and conformational study of two L-prolyl-L-leucyl-glycinamide analogues with a reduced peptide bond

The peptide bond between Pro-Leu or Leu-Gly in Pro-Leu-Gly-NH2 was replaced by a CH2-NH function. The 1H and 13C n.m.r. studies demonstrated that HCl X Pro-Leu psi (CH2-NH)Gly-NH2 10 adopted a conformation in DMSO that is similar to the previously postulated beta-turn for the natural hormone. This was not the case for the other analogue. In vivo tests on 10 revealed an activity approximately equal to the natural compound and an increased toxicity.     

Further evidence for a C-terminal structural motif in CCK2 receptor active peptide hormones

A comparison of the conformational characteristics of the related hormones [Nle(15)] gastrin-17 and [Tyr(9)-SO(3)] cholecystokinin-15, in membrane-mimetic solutions of dodecylphosphocholine micelles and water, was undertaken using NMR spectroscopy to investigate the possibility of a structural motif responsible for the two hormones common ability to stimulate the CCK(2) receptor. Distance geometry calculations and NOE-restrained molecular dynamics simulations in biphasic solvent boxes of decane and water pointed to the two peptides adopting near identical helical C-terminal configurations, which extended one residue further than their shared pentapeptide sequence of Gly-Trp-Met-Asp-Phe-NH(2). The C-terminal conformation of [Nle(15)] gastrin-17 contained a short alpha-helix spanning the Ala(11)-Trp(14) sequence and an inverse gamma-turn centered on Nle(15) while that of [Tyr(9)-SO(3)] cholecystokinin-15 contained a short 3(10) helix spanning its Met(10) to Met(13) sequence and an inverse gamma-turn centered on Asp(14). Significantly, both the C-terminal helices were found to terminate in type I beta-turns spanning the homologous Gly-Trp-Met-Asp sequences. This finding supports the hypothesis that this structural motif is a necessary condition for CCK(2) receptor activation given that both gastrin and cholecystokinin have been established to follow a membrane-associated pathway to receptor recognition and activation. Comparison of the conformations for the non-homologous C-terminal tyrosyl residues of [Nle(15)] gastrin-17 and [Tyr(9)-SO(3)] cholecystokinin-15 found that they lie on opposite faces of the conserved C-terminal helices. The positioning of this tyrosyl residue is known to be essential for CCK(1) activity and non-essential for CCK(2) activity, pointing to it as a possible differentiator in CCK(1)/CCK(2) receptor selection. The different tyrosyl orientations were retained in molecular models for the [Nle(15)] gastrin-17/CCK(2) receptor and [Tyr(9)-SO(3)] cholecystokinin-15/CCK(1) receptor complexes, highlighting the role of this residue as a likely CCK(1)/CCK(2) receptor differentiator.     

Relative stability of alpha-melanotropin and related analogues to rat brain homogenates

alpha-Melanotropin (alpha-MSH) retains less than 1% of its original activity after a 60 min incubation with 10% rat brain homogenate. [Nle4,D-Phe7]-alpha-MSH is nonbiodegradable in rat serum (240 min incubation) and still maintains 10% of its original activity in 10% rat brain homogenate (240 min incubation). The related fragment analogue, Ac-[Nle4,D-Phe7]-alpha-MSH4-10-NH2, retains 50% of its activity after a 240 min incubation in rat brain homogenate, whereas Ac-[Nle4,D-Phe7]-alpha-MSH4-11-NH2 is totally resistant to inactivation by rat brain homogenate. Both [Nle4,D-Phe7]-fragments are resistant to degradation by rat serum, but [Nle4]-alpha-MSH, Ac-[Nle4]-alpha-MSH4-10-NH2 and Ac-[Nle4]-alpha-MSH4-11-NH2 are rapidly inactivated under both conditions. The cyclic melanotropin, [Cys4,Cys10]-alpha-MSH, is inactivated in rat brain homogenate as is the shorter Ac-[Cys4,Cys10]-alpha-MSH4-10-NH2 analogue, but neither cyclic melanotropin is inactivated upon incubation in serum from rats. Ac-[Cys4,D-Phe7,Cys10]-alpha-MSH4-10-NH2 is resistant to inactivation by either rat serum or a brain homogenate. Some of these melanotropin analogues may provide useful probes for the localization and characterization of putative melanotropin receptors in both the central nervous system and peripheral tissues.     

Analogs of oxytocin containing a modified peptide bond

Analogs of deamino-oxytocin and deamino-oxypressin containing a CH2-NH group instead of an amide bond between positions 8 and 9 were synthesized. All tested compounds exhibit significantly lowered biological activities.     

Structure-function relationship studies of bovine parathyroid hormone [bPTH(1-34)] analogues containing alpha-amino-iso-butyric acid (Aib) residues

The N-terminal 1-34 fragments of the parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHrP) elicit the full spectrum of bone-related biological activities of the intact native sequences. It has been suggested that the structural elements essential for bioactivity are two helical segments located at the N-terminal and C-terminal sequences, connected by hinges or flexible points around positions 12 and 19. In order to assess the relevance of the local conformation around Gly(12) upon biological function, we synthesized and characterized the following PTH(1-34) analogues containing Aib residues: (I) A-V-S-E-I-Q-F-nL-H-N-Aib-G-K-H-L-S-S-nL-E-R-V-E-Nal-L-R-K-K-L-Q-D-V-H-N-Y-NH(2) ([Nle(8,18), Aib(11), Nal(23),Tyr(34)]bPTH(1-34)-NH(2)); (II) A-V-S-E-I-Q-F-nL-H-N-L-Aib-K-H-L-S-S-nL-E-R-V-E-Nal-L-R-K-K-L-Q-D-V-H-N-Y-NH(2) ([Nle(8,18), Aib(12),Nal(23),Tyr(34)]bPTH(1-34)-NH(2)); (III) A-V-S-E-I-Q-F-nL-H-N-L-G-Aib-H-L-S-S-nL-E-R-V-E-Nal-L-R-K-K-L-Q-D-V-H-N-Y-NH(2) ([Nle(8,18), Aib(13), Nal(23),Tyr(34)]bPTH(1-34)-NH(2)); (IV) A-V-S-E-I-Q-F-nL-H-N-Aib-Aib-K-H-L-S-S-nL-E-R-V-E-Nal-L-R-K-K-L-Q-D-V-H-N-YNH(2) ([Nle(8,18), Aib(11,12), Nal(23),Tyr(34)]bPTH(1-34)-NH(2)); (V) A-V-S-E-I-Q-F-nL-H-N-L-Aib-Aib-H-L-S-S-nL-E-R-V-E-Nal-L-R-K-K-L-Q-D-V-H-N-Y-NH(2) ([Nle(8,18), Aib(12,13),Nal(23),Tyr(34)]bPTH(1-34)-NH(2)). (nL= Nle; Nal= L-(2-naphthyl)-alanine; Aib= alpha-amino-isobutyric acid.) The introduction of Aib residues at position 11 in analogue I or at positions 11 and 12 in analogue IV resulted in a 5-20-fold lower efficacy and a substantial loss of binding affinity compared to the parent compound [Nle(8,18), Nal(23),Tyr(34)]bPTH(1-34)-NH(2). Both binding affinity and adenylyl cyclase stimulation activity are largely restored when the Aib residues are introduced at position 12 in analogue II, 13 in analogue III, and 12-13 in analogue V. The conformational properties of the analogues in aqueous solution containing dodecylphosphocholine micelles were studied by CD, two-dimensional (2D) NMR and computer simulations. The results indicated the presence of two helical segments in all analogues, located at the N-terminal and C-terminal sequences. Insertion of Aib residues at positions 12 and 13, or of Aib dyads at positions 11-12 and 12-13, enhances the stability of the N-terminal helix of all analogues. In all analogues the Aib residues are included in the helical segments. These results confirmed the importance of the helical structure in the N-terminal activation domain, as well as of the presence of the Leu(11) hydrophobic side chain in the native sequence, for PTH-like bioactivity.     

Mutation of asparagine 229 to aspartate in thymidylate synthase converts the enzyme to a deoxycytidylate methylase.

The conserved Asn 229 of thymidylate synthase (TS) forms a cyclic hydrogen bond network with the 3-NH and 4-O of the nucleotide substrate dUMP. The Asn 229 to Asp mutant of Lactobacillus casei thymidylate synthase (TS N229D) has been prepared, purified, and investigated. Steady-state kinetic parameters of TS N229D show 3.5- and 10-fold increases in the Km values of CH2H4folate and dUMP, respectively, and a 1000-fold decrease in kcat. Most important, the Asp 229 mutation changes the substrate specificity of TS to an enzyme which recognizes and methylates dCMP in preference to dUMP. With TS N229D the Km for dCMP is bout 3-fold higher than for dUMP, and the Km for CH2H4folate is increased about 5-fold; however, the kcat for dCMP methylation is 120-fold higher than that for dUMP methylation. Specificity for dCMP versus dUMP, as measured by kcat/Km, changes from negligible with wild-type TS to about a 40-fold increase with TS N229D. TS N229D reacts with CH2H4folate and FdUMP or FdCMP to form ternary complexes which are analogous to the TS-FdUMP-CH2H4folate complex. From what is known of the mechanism and structure of TS, the dramatic change in substrate specificity of TS N229D is proposed to involve a hydrogen bond network between Asp 229 and the 3-N and 4-NH2 of the cytosine heterocycle, causing protonation of the 3-N and stabilization of a reactive imino tautomer. A similar mechanism is proposed for related enzymes which catalyze one-carbon transfers to cytosine heterocycles.     

Probing the Ser-Ser-Lys catalytic triad mechanism of peptide amidase: computational studies of the ground state, transition state, and intermediate

Peptide amidase (Pam), a hydrolytic enzyme that belongs to the amidase signature (AS) family, selectively catalyzes the hydrolysis of the C-terminal amide bond (CO-NH(2)) of peptides. The recent availability of the X-ray structures of Pam, fatty acid amide hydrolase, and malonamidase E2 has led to the proposal of a novel Ser-Ser-Lys catalytic triad mechanism for the amide hydrolysis by the AS enzymes. The molecular dynamics (MD) simulations using the CHARMM force field were performed to explore the catalytic mechanism of Pam. The 1.8 A X-ray crystal structure of Pam in complex with the amide analogue of chymostatin was chosen for the initial coordinates for the MD simulations. The five systems that were investigated are as follows: (i) enzyme.substrate with Lys123-NH(2), (ii) enzyme.substrate with Lys123-NH(3)(+), (iii) enzyme.substrate with Lys123-NH(3)(+) and Ser226-O(-), (iv) enzyme.transition state, and (v) enzyme.tetrahedral intermediate. Our data support the presence of the hydrogen bonding network among the catalytic triad residues, Ser226, Ser202, and Lys123, where Ser226 acts as the nucleophile and Ser202 bridges Ser226 and Lys123. The MD simulation supports the catalytic role of the crystallographic waters, Wat1 and Wat2. In all the systems that have been studied, the backbone amide nitrogens of Asp224 and Thr223 create an oxyanion hole by hydrogen bonding to the terminal amide oxygen of the substrate, and stabilize the oxyanion tetrahedral intermediate. The results from both our computational investigation and previously published experimental pH profile support two mechanisms. In a mechanism that is relevant at lower pH, the Lys123-NH(3)(+)-Ser202 dyad provides structural support to the catalytic residue Ser226, which in turn carries out a nucleophilic attack at the substrate amide carbonyl in concert with Wat1-mediated deprotonation and stabilization of the tetrahedral transition state by the oxyanion hole. In the mechanism operating at higher pH, the Lys123-NH(2)-Ser202 catalytic dyad acts as a general base to assist addition of Ser226 to the substrate amide carbonyl. The results from the MD simulation of the tetrahedral intermediate state show that both Ser202 and Lys123 are possible candidates for protonation of the leaving group, NH(2), to form the acyl-enzyme intermediate.     

Purification and characterization of a receptor for human parathyroid hormone and parathyroid hormone-related peptide

The human parathyroid hormone (PTH) receptor (hPTH1R), containing a 9-amino acid sequence of rhodopsin at its C terminus, was transiently expressed in COS-7 cells and solubilized with 0.25% n-dodecyl maltoside. Approximately 18 microg of hPTH1R were purified to homogeneity per mg of crude membranes by single-step affinity chromatography using 1D4, a monoclonal antibody to a rhodopsin epitope. The N terminus of the hPTH1R is Tyr(23), consistent with removal of the 22-amino acid signal peptide. Comparisons of hPTH1R by quantitative immunoblotting and Scatchard analysis revealed that 75% of the receptors in membrane preparations were functional; there was little, if any, loss of functional receptors during purification. The binding affinity of the purified hPTH1R was slightly lower than membrane-embedded hPTH1R (K(d) = 16.5 +/- 1.3 versus 11.9 +/- 1.9 nm), and the purified receptors bound rat [Nle(8,21),Tyr(34)]PTH-(1-34)-NH(2) (PTH-(1-34)), and rat [Ile(5),Trp(23),Tyr(36)]PTHrP-(5-36)-NH(2) with indistinguishable affinity. Maximal displacement of (125)I-PTH-(1-34) binding by rat [alpha-aminoisobutyric acid (Aib)(1,3),Nle(8),Gln(10),Har(11),Ala(12),Trp(14),Arg(19),Tyr(21)]PTH-(1-21)-NH(2) and rat [Aib(1,3),Gln(10),Har(11),Ala(12),Trp(14)]PTH-(1-14)-NH(2) of 80 and 10%, respectively, indicates that both N-terminal and juxtamembrane ligand binding determinants are functional in the purified hPTH1R. Finally, PTH stimulated [(35)S]GTP gamma S incorporation into G alpha(s) in a time- and dose-dependent manner, when recombinant hPTH1R, G alpha(s)-, and beta gamma-subunits were reconstituted in phospholipid vesicles. The methods described will enable structural studies of the hPTH1R, and they provide an efficient and general technique to purify proteins, particularly those of the class II G protein-coupled receptor family.