Sonic hedgehog delivered by an adeno-associated virus protects dopaminergic neurones against 6-OHDA toxicity in the rat.

Direct intracerebral administration of sonic hedgehog (SHH) reduces 6-OHDA and MPTP toxicity to nigral dopaminergic cells in rats and primates. To determine whether transfection of the DNA sequence for SHH using viral vectors also protects against 6-OHDA toxicity, a type 2 adeno- associated virus (AAV) incorporating 600 base pairs of N-terminal SHH DNA was generated to induce SHH expression in rat striatum.AAV-SHH was injected into the striatum, 3 weeks prior to the initiation of an unilateral partial 6-OHDA nigro-striatal lesion. Animals receiving 4x10(7) viral particles of AAV-SHH showed a reduction in (+)-amphetamine induced ipsilateral turning over 4 weeks, when compared to animals receiving vehicle or a LacZ encoding vector. Following vehicle or AAV-LacZ administration, 6-OHDA caused a marked loss of striatal dopamine content and nigral tyrosine hydroxylase (TH) immunopositive cells. Following treatment with 4x10(7) viral particles of AAV-SHH the loss of striatal dopamine content was reduced and there was marked preservation of nigral dopaminergic cells. However, administration of 4x10(8) particles of AAV-SHH did not cause a significant change in (+)-amphetamine-induced rotation, striatal dopamine levels or the number of nigral TH immunoreactive cells following 6-OHDA lesioning compared to vehicle or AAV-LacZ treated animals. The results show that SHH delivered via a viral vector can protect dopaminergic neurons against 6-OHDA toxicity and suggest that this could be developed into a novel treatment for PD. However, the effects maybe dose limited due to uncoupling of hedgehog receptor signalling at higher levels of SHH expression.

Intrastriatal sonic hedgehog injection increases Patched transcript levels in the adult rat subventricular zone.

The morphogen sonic hedgehog (Shh) is implicated in neural tissue patterning and the growth of brain structures during embryogenesis and postnatal development and is also present in the adult brain. Shh signals through interaction with the tumour suppressor Patched (Ptc). This receptor for Shh is associated with Smoothened (Smo), a protein with high homology to the G-protein coupled receptors. However, little is known about the transduction mechanisms implicated in Shh signalling in the adult brain. The study described here shows that injection of aminoterminal myristoylated Shh (myrShhN) into the adult rat striatum robustly increases the levels of Ptc transcripts in selective brain areas including the subventricular zone (SVZ). The adult SVZ contains cell progenitors, which can proliferate and differentiate into new neurons and glia. In the myrShhN injected animals, proliferation and differentiation of these SVZ precursor cells were not affected as demonstrated by BrdU incorporation and immunohistochemistry performed with specific antibodies for nestin (uncommitted neural progenitors), PSA-NCAM (migrating neuroblasts) or GFAP (astrocytes). Together with the presence of Smo expressing cells and amino-terminal Shh (ShhN) protein in SVZ area of untreated animals, the data presented here supports the hypothesis that the Shh pathway may be activated in the adult brain, and that a niche for Shh signalling exists within the adult SVZ.

Behavioural and immunohistochemical changes following supranigral administration of sonic hedgehog in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated common marmosets.

Sonic hedgehog (SHH) has trophic actions on dopaminergic cell cultures and protects them from MPP(+) toxicity but its in vivo actions have not been explored. We now investigate the effects of unilateral supranigral administration of SHH on nigro-striatal function in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated common marmosets. SHH (0.1 or 1.0 microg) or vehicle was stereotaxically injected into the region of the right substantia nigra twice with an interval of 5 weeks between administrations. The first or second administration of low dose SHH (0.1 microg) did not significantly improve motor disability or locomotor activity compared to time-matched vehicle-treated animals. There was, however, an approximately 30% improvement in both motor disability and locomotor activity following the first administration of high dose SHH (1.0 microg). No further improvements occurred following the second high dose SHH treatment. Acute oral administration of L-3,4-dihydroxyphenylalanine (L-DOPA) produced a smaller increase in locomotor activity and greater reversal of motor disability in animals treated with SHH than occurred in vehicle-treated common marmosets. In the substantia nigra pars compacta, ipsilateral to SHH administration, the number of tyrosine hydroxylase-positive neurones was increased by 21% (P > 0.05) and 57% (P < 0.05) in low and high dose SHH groups respectively compared to the untreated contralateral hemisphere. There was no difference in the number of glial fibrillary acidic protein-positive cells. SHH may improve nigro-striatal function by restoring tyrosine hydroxylase positivity. This is reflected by an improvement in basal disability and a reduction in the lesion-induced response to L-DOPA.

Enhanced potency of human Sonic hedgehog by hydrophobic modification.

Post-translational modifications of the developmental signaling protein Sonic hedgehog (Shh) by a long-chain fatty acid at the N-terminus and cholesterol at the C-terminus greatly activate the protein in a cell-based signaling assay. To investigate the structural determinants of this activation phenomenon, hydrophobic and hydrophilic moieties have been introduced by chemical and mutagenic methods to the soluble N-terminal signaling domain of Shh and tested in both in vitro and in vivo assays. A wide variety of hydrophobic modifications increased the potency of Shh when added at the N-terminus of the protein, ranging from long-chain fatty acids to hydrophobic amino acids, with EC(50) values from 99 nM for the unmodified protein to 0.6 nM for the myristoylated form. The N-myristoylated Shh was as active as the natural form having both N- and C-terminal modifications. The degree of activation appears to correlate with the hydrophobicity of the modification rather than any specific chemical feature of the adduct; moreover, substitution with hydrophilic moieties decreased activity. Hydrophobic modifications at the C-terminus of Shh resulted in only a 2-3-fold increase in activity, and no activation was found with hydrophobic modification at other surface positions. The N-terminal modifications did not appear to alter the binding affinity of the Shh protein for the transfected receptor protein, Patched, and had no apparent effect on structure as measured by circular dichroism, thermal denaturation, and size determination. Activation of Desert Hh through modification of its N-terminus was also observed, suggesting that this is a common feature of Hh proteins.

Mapping sonic hedgehog-receptor interactions by steric interference.

We have defined regions in the Sonic hedgehog (Shh) molecule that are important for Patched (Ptc) receptor binding by targeting selected surface amino acid residues with probes of diverse sizes and shapes and assessing the effects of these modifications on function. Eleven amino acid residues that surround the surface of the protein were chosen for these studies and mutated to cysteine residues. These cysteines were then selectively modified with thiol-specific probes, and the modified proteins were tested for hedgehog receptor binding activity and their ability to induce differentiation of C3H10T1/2 cells into osteoblasts. Based on these analyses, approximately one-third of the Shh surface can be modified without effect on function regardless of the size of the attachment. These sites are located near to where the C terminus protrudes from the surface of the protein. All other sites were sensitive to modification, indicating that the interaction of Shh with its primary receptor Ptc is mediated over a large surface of the Shh protein. For sites Asn-50 and Ser-156, function was lost with the smallest of the probes tested, indicating that these residues are in close proximity to the Ptc-binding site. The epitope for the neutralizing mAb 5E1 mapped to a close but distinct region of the structure. The structure-activity data provide a unique view of the interactions between Shh and Ptc that is not readily attainable by conventional mapping strategies.

Zinc-dependent structural stability of human Sonic hedgehog.

The role of the zinc site in the N-terminal fragment of human Sonic hedgehog (ShhN) was explored by comparing the biophysical and functional properties of wild-type ShhN with those of mutants in which the zinc-coordinating residues H140, D147, and H182, or E176 which interacts with the metal ion via a bridging water molecule, were mutated to alanine. The wild-type and E176A mutant proteins retained 1 mol of zinc/mol of protein after extensive dialysis, whereas the H140A and D147A mutants retained only 0.03 and 0.05 mol of zinc/mol of protein, respectively. Assay of the wild-type and mutant proteins in two activity assays indicated that the wild-type and E176A mutant proteins had similar activity, whereas the H140A and D147A mutants were significantly less active. These assays also indicated that the H140A and D147A mutants were susceptible to proteolysis. CD, fluorescence, and (1)H NMR spectra of the H140A, D147A, and E176A mutants measured at 20 or 25 degrees C were very similar to those observed for wild-type ShhN. However, CD measurements at 37 degrees C showed evidence of some structural differences in the H140A and D147A mutants. Guanidine hydrochloride (GuHCl) denaturation studies revealed that the loss of zinc from the H140A and D147A mutants destabilized the folded proteins by approximately 3.5 kcal/mol, comparable to the effect of removing zinc from wild-type ShhN by treatment with EDTA. Thermal melting curves of wild-type ShhN gave a single unfolding transition with a midpoint T(m) of approximately 59 degrees C, whereas both the H140A and D147A mutants displayed two distinct transitions with T(m) values of 37-38 and 52-54 degrees C, similar to that observed for EDTA-treated wild-type ShhN. Addition of zinc to the H140A and D147A mutants resulted in a partial restoration of stability against thermal and GuHCl denaturation. The ability of these mutants to bind zinc was confirmed using a fluorescence-based binding assay that indicated that they bound zinc with K(d) values of approximately 1.6 and approximately 15 nM, respectively, as compared to a value of

Identification of a palmitic acid-modified form of human Sonic hedgehog.

During hedgehog biosynthesis, autocatalytic processing produces a lipid-modified amino-terminal fragment (residues 24-197 in the human Sonic hedgehog sequence) that is responsible for all known hedgehog signaling activity and that is highly conserved evolutionarily. Published in vitro biochemical studies using Drosophila hedgehog identified the membrane anchor as a cholesterol, and localized the site of attachment to the COOH terminus of the fragment. We have expressed full-length human Sonic hedgehog in insect and in mammalian cells and determined by mass spectrometry that, in addition to cholesterol, the human hedgehog protein is palmitoylated. Peptide mapping and sequencing data indicate that the palmitoyl group is attached to the NH2 terminus of the protein on the alpha-amino group of Cys-24. Cell-free palmitoylation studies demonstrate that radioactive palmitic acid is readily incorporated into wild type Sonic hedgehog, but not into variant forms lacking the Cys-24 attachment site. The lipid-tethered forms of hedgehog showed about a 30-fold increase in potency over unmodified soluble hedgehog in a cell- based (C3H10T1/2 alkaline phosphatase induction) assay, suggesting that the lipid tether plays an important role in hedgehog function. The observation that an extracellular protein such as Shh is palmitoylated is highly unusual and further adds to the complex nature of this protein.

Motilin and OHM-11526 activate a calcium current in human and canine jejunal circular smooth muscle.

Motilin is a potent agonist for gastrointestinal smooth muscle contraction and has been proposed to regulate the onset of phase III of the migrating motor complex in dogs and humans. The effects of motilin and OHM-11526, a motilin antagonist in rabbit smooth muscle strips, were examined in isolated canine and human jejunal circular smooth muscle cells using whole cell patch-clamp techniques with Ba2+ as the charge carrier. Effects of both drugs on inward current through L-type Ca2+ channels (ICaL) in both canine and human cells were first observed at 10(-3) M. At 10(-6) M, motilin increased ICaL in canine and human jejunal circular smooth muscle cells by 43 +/- 20 and 45 +/- 11%, respectively, and OHM-11526 increased ICaL by 54 +/- 8 and 54 +/- 14%, respectively. The increase in inward current was blocked by nifedipine and by guanosine 5'-O-(2-thiodiphosphate) but not by pertussis toxin. Washout of both drugs resulted in a further increase in ICaL. These data suggest that both motilin and OHM-11526 activate and ICaL in human and canine jejunal circular smooth muscle cells through a G protein-coupled mechanism.

Antagonistic properties of [Phe3,Leu13]porcine motilin.

We describe the antagonistic properties due to the replacement of Pro3 by phenylalanine in porcine motilin. The analogue, [Phe3,Leu13] porcine motilin (OHM-11526), displaces iodinated [Nle13]porcine motilin bound to a homogenate of rabbit antral smooth muscle tissue. The dissociation constant (pKd) was 9.26 +/- 0.04, versus 9.11 +/- 0.01 for motilin and 8.24 +/- 0.06 for ANQ-11125, the (1-14) fragment of OHM-11526. The Hill coefficient was close to one and Schild plot analysis confirmed the competitive nature of the interaction. In the tissue bath OHM-11526 was unable to induce contractions of segments of rabbit duodenum. At a concentration of 10(-6) M, OHM-11526 was unable to induce contractions of segments of rabbit duodenum. At a concentration of 10(-6) M, OHM-11526 inhibited the effect of maximally effective doses of porcine motilin and of the erythromycin derivative, EM-523, but was without effect on contractions induced by acetylcholine, substance P and serotonin. Increasing doses of OHM-11526 shifted the dose-response curves of motilin and EM-523 to the right, but caused a depression of the maximal response as well. From the motilin curves, and assuming a dual competitive and non-competitive interaction, the pA2 was 7.79 +/- 0.08, the pD'2 6.91 +/- 0.08. The EM-523 curves yielded comparable data (pA2 = 8.10 +/- 0.12 and pD'2 = 7.06 +/- 0.13). OHM-11526 also blocked the motilin responses observed with smooth muscle strips from the rabbit and human antrum. However, in a preparation of the chicken small intestine, OHM-11526 was a full agonist with a potency (pD2 = 6.84) comparable to that of porcine motilin (pD2 = 6.71). Our data confirm the interaction of motilides with the motilin receptor. Due to its increased affinity for the motilin receptor, OHM-11526 will be a valuable took for studying the physiology of motilin and the pharmacology of motilin and motilides.

The motilin antagonist ANQ-11125 blocks motilide-induced contractions in vitro in the rabbit.

Studies on the physiological role of motilin, and more recently, on the relationship between motilin and erythromycin A, have been hampered by the lack of antagonists. We now have discovered such a compound. ANQ-11125 displaces motilin bound to an homogenate of rabbit antral smooth muscle tissue. The dissociation constant (pKd) was 8.16 +/- 0.10. However, ANQ-11125 did not induce contractions of segments of rabbit duodenum, except at high concentrations. In the presence of 1 microM ANQ-11125 the dose response curves of erythromycin-A, De(N-methyl)-N-ethyl-8,9 anhydroerythromycin A 6,9-hemiacetal and motilin were shifted about one log unit to the right, but the responses to ACh and Substance P were unaffected. Schild-analysis showed the competitive nature of the interaction and allowed the calculation of the pA2: 7.03 +/- 0.05 (motilin curves) and 7.55 +/- 0.06 (EM-523 curves). This is the first report of a motilin antagonist. Its properties definitively prove that motilides are motilin agonists.

Solution structure of the calcium channel antagonist omega-conotoxin GVIA.

The three-dimensional solution structure is reported for omega-conotoxin GVIA, which is a potent inhibitor of presynaptic calcium channels in vertebrate neuromuscular junctions. Structures were generated by a hybrid distance geometry and restrained molecular dynamics approach using interproton distance, torsion angle, and hydrogen-bonding constraints derived from 1H NMR data. Conformations of GVIA with low constraint violations converged to a common peptide fold. The secondary structure in the peptide is an antiparallel triple-stranded beta-sheet containing a beta-hairpin and three tight turns. The NMR data are consistent with the region of the peptide from residues S9 to C16 being more dynamic than the rest of the peptide. The peptide has an amphiphilic structure with a positively charged hydrophilic side and an opposite side that contains a small hydrophobic region. Residues that are thought to be important in binding and function are located on the hydrophilic face of the peptide.

D-amino acid and alanine scans of the bioactive portion of porcine motilin.

A recent systematic study of porcine motilin fragments has clearly shown that biological activity resides in the amino-terminal end. The amino-terminal tetradecapeptide retains more than 90% of the potency of the full molecule. We now examined the effect of replacement of residues 1 through 11 by either their D-isomer or by alanine in [Leu13]pMOT(1-14). Peptides were synthesized using Fmoc solid phase methodology, purified by HPLC, and assayed for their ability to displace bound motilin (rabbit antral smooth muscle homogenate) and to induce contractions (isolated rabbit duodenal segments). The negative logarithm of the concentration displacing 50% of the tracer (pIC50), or producing 50% of the maximal contractile response (pEC50), was determined. All compounds were still full agonists. A reduction in potency of more than two log units was seen for the compounds in which residues 1 (Phe), 4 (Ile), and 7 (Tyr) were replaced by Ala and residues 3 (Pro), 4 (Ile), and 6 (Thr) by their D-isomer. The largest drop was noted for the analogs substituted at position 4. For all compounds there was an almost perfect correlation between the pIC50 and the pEC50 values (r = 0.96), although the pEC50 was consistently smaller. These results show that the biological activity of motilin is mainly determined by the first seven residues. The pharmacophore consists of the aromatic rings from Phe1 and Tyr7 and the aliphatic side chains from Val2 and Ile4. Pro3, Phe5, and Thr6 may stabilize the bioactive conformation.

Synthesis and in vitro evaluation of [Leu13]porcine motilin fragments.

Several peptide fragments representing N-terminal, C-terminal, and internal sequences of [Leu13]porcine motilin ([Leu13]pMOT) were synthesized using Fmoc solid phase methodology. Peptides were assayed for motilin receptor binding activity in a rabbit antrum smooth muscle preparation and for stimulation of contractile activity in segments of rabbit duodenum. In vitro activity was directly correlated with motilin receptor binding affinity for all [Leu13]pMOT fragments examined. N-Terminal fragments of just over half the length of the native peptide are nearly equipotent as full-length motilin. These results suggest that the N-terminal segment, together with residues from the mid-portion of the molecule, constitutes the bioactive portion of pMOT. The C-terminal segment, in contrast, contributes little to receptor binding affinity or in vitro activity.

Molecular modeling of 5-HT3 receptor ligands.

Ligands of various chemical classes (e.g., indoles, indazoles, benzamides, carbazoles, and quinolines) have demonstrated high affinity for the 5-HT3 receptor in radiolabeled ligand-binding studies, and have shown 5-HT3 receptor antagonistic activity in functional assays which utilize the excitatory effects of 5-HT on enteric neurons and autonomic afferents. Several 5-HT3 antagonists are currently being evaluated for potential use in the treatment of migraine, schizophrenia, and anxiety, and a few have already demonstrated high efficacy as antiemetics in cancer chemotherapy. The purpose of this presentation is to highlight the significant structure-affinity relationships (SAFIR) and common geometrical features among 5-HT3 receptor ligands, and to describe the three-dimensional pharmacophore for the 5-HT3 recognition site derived from computational techniques. The chemical template containing the recognition elements (functional groups) for the 5-HT3 receptor are: an aromatic or heteroaromatic ring system, a coplanar carbonyl group, and a nitrogen center, interrelated by well-defined distances. Two "binding shapes" or "active shapes" for 5-HT3 ligands have been identified from detailed conformational analyses.

Capillary zone electrophoresis studies of motilin peptides. Effects of charge, hydrophobicity, secondary structure and length.

Motilin is a gut hormone, which is involved in gastrointestinal motility. Capillary electrophoresis studies were made on 24 peptides that are N-terminal, C-terminal or internal fragments of motilin. The isoelectric point, total charge and hydrophobicity were calculated for all of the peptides. The effects of buffers and pH on migration time and resolution were studied. These included citrate buffer, pH 2.5; phosphate buffer, pH 7.0 and borate buffer, pH 10.0. A capillary zone electrophoresis method was developed to resolve 14 of the motilin peptides. Secondary structure predictions were made using the Chou-Fasman method. Circular dichroism spectra were collected to confirm presence of alpha-helix in several fragments. Effects of charge, hydrophobicity, secondary structure and length of the motilin fragments on migration time were studied.

Solution structures of alpha-conotoxin G1 determined by two-dimensional NMR spectroscopy.

Two-dimensional NMR data have been used to generate solution structures of alpha-conotoxin G1, a potent peptide antagonist of the acetylcholine receptor. Structural information was obtained in the form of proton-proton internuclear distance constraints, and initial structures were produced with a distance geometry algorithm. Energetically more favorable structures were generated by using the distance geometry structures as input for a constrained energy minimization program. The results of both of these calculations indicate that the overall backbone conformation of the molecule is well-defined by the NMR data whereas the side-chain conformations are generally less well-defined. The main structural features derived from the NMR data were the presence of tight turns centered on residues Pro5 and Arg9. The solution structures are compared with previous proposed models of conotoxin G1, and the NMR data are interpreted in conjunction with chemical modification studies and structural properties of other antagonists of the acetylcholine receptor to gain insight into structure-activity relationships in these peptide toxins.

High-performance liquid chromatographic separation of peptide and amino acid stereoisomers.

Several high-performance liquid chromatographic (HPLC) methods are described for separation of peptide stereoisomers which are not well resolved by traditional reversed-phase chromatography. These chiral HPLC methods include investigations with a beta-cyclodextrin column, a Pirkle D-Phenyl Glycine column and a Chiral-Pak WH column. A method based on derivatization of dipeptides with a chiral reagent, N-acetyl-L-cysteine and o-phthalaldehyde, is also discussed. A series of linear and cyclic dipeptides and modified amino acids were chromatographed on the four systems. Resolution varied for the four different systems depending on the types of compounds that were chromatographed.

Rapid-scanning cryospectroscopy of enzyme-substrate-inhibitor complexes of cobalt carboxypeptidase A.

Rapid-scanning cryospectroscopy of cobalt(II)-substituted carboxypeptidase A serves to identify and characterize ternary enzyme-substrate-inhibitor (IES) complexes formed by the interaction between the enzyme, a peptide substrate, and a noncompetitive inhibitor. A cobalt absorption spectrum distinct from any induced by peptide or inhibitor alone signals formation of the IES complex. Tight-binding noncompetitive inhibitors containing an aromatic ring, e.g., beta-phenylpropionate, cause the IES complex to form much more slowly than simple binary complexes of the enzyme with either peptide or inhibitor. An inhibitor such as acetate, which binds more weakly and is less bulky, permits the IES complex to form relatively quickly. Remarkably, the cobalt spectra of the IES complexes match those previously found for the steady-state ester (depsipeptide) intermediates. Chemical quenching studies have demonstrated that in these ester intermediates the scissile bond is broken [Galdes, A., Auld, D. S., & Vallee, B. L. (1986) Biochemistry 25, 646-651]. This finding, in conjunction with the present studies, implies that a peptide and a noncompetitive inhibitor of its hydrolysis occupy the same binding loci as the hydrolytic products of a depsipeptide and further indicates that breakdown of an enzyme-biproduct complex is rate-determining for the turnover of depsipeptides.

Hydrolysis of peptides by carboxypeptidase A: equilibrium trapping of the ES2 intermediate.

The cobalt absorption and electron paramagnetic resonance (EPR) spectra of cobalt carboxypeptidase undergo unique variations on formation of catalytic peptide and ester intermediates as previously recorded in cryoenzymologic experiments employing rapid-scanning spectroscopy and cryotrapping [Geoghegan, K. F., Galdes, A., Martinelli, R. A., Holmquist, B., Auld, D.S., & Vallee, B. L. (1983) Biochemistry 22, 2255-2262]. We here describe a means of stabilizing these intermediates, which we have termed "equilibrium trapping". It allows peptide intermediates to be observed for longer periods (much greater than 1 min) at ambient as well as subzero temperatures. The reaction intermediate with the rapidly turned over peptide substrate Dns-Ala-Ala-Phe is trapped when the cobalt enzyme (greater than 10 microM) has catalyzed the attainment of chemical equilibrium between high concentrations of the hydrolysis products Dns-Ala-Ala, 10 mM, and L-phenylalanine, 50 mM, and the product of their coupling Dns-Ala-Ala-Phe. Under these conditions, Dns-Ala-Ala-Phe is present in the equilibrated substrate-product reaction mixture at a level that exceeds the one predicted on the basis of K'eq for hydrolysis of this substrate and is close to the enzyme concentration. Other pairs of peptide hydrolysis products yield similar results. Visible absorption and EPR spectra of the cobalt enzyme show that the synthesized peptide binds to the active site in the mode previously recognized as the ES2 catalytic intermediate in peptide hydrolysis. Equilibrium trapping of the ES2 intermediate allows analysis of its physicochemical properties by methods that could not be employed readily under cryoenzymological conditions, e.g., circular dichroic and magnetic circular dichroic spectra.(ABSTRACT TRUNCATED AT 250 WORDS)

Elucidation of the chemical nature of the steady-state intermediates in the mechanism of carboxypeptidase A.

Cryospectrokinetic studies of zinc and cobalt carboxypeptidase A disclosed two intermediates in the hydrolysis of both peptides and depsipeptides and furnished all the rate and equilibrium constants for the reaction scheme E + S in equilibrium ES1 in equilibrium ES2---E + P [Auld, D. S., Galdes, A., Geoghegan, K. F., Holmquist, B., Martinelli, R. A., & Vallee, B. L. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 5041-5045]. Since the ES2 intermediate is the predominate enzyme species present at steady state, its chemical nature is deducible from subzero chemical quench studies done after steady state is established. Extrapolation of the product concentration to zero time, [P0], measures the concentration of the enzyme species in which bond cleavage has occurred. For peptides, the [P0]values are zero, indicating that no product is generated prior to turnover and therefore the ES2 intermediate involves a complex between enzyme and intact peptide substrate. For depsipeptides, [P0] values are 1 mol of produce per mole of enzyme over the entire temperature range -20 to -50 degrees C, indicating cleavage of the ester bond occurs prior to the rate-limiting step so that ES2 is more properly denoted by EP1P2, where P1 and P2 are the substrates for the reverse reaction. The rate-limiting step for depsipeptides thus involves release of the products which may occur directly or through a mandatory conformational change followed by rapid product release.