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Passive peptide transport across lipid membranes is governed by the energetics of partitioning into the ordered chain interior coupled with the rate of diffusion across this region. A hydrophobicity scale for peptide transfer into the barrier region of membranes derived from permeability coefficients would be useful to predict passive permeation of peptides across biomembranes and for determining the thermodynamics of peptide/protein insertion into the membrane interior. This study reports transport rates across large unilamellar vesicles (LUVs) composed of egg lecithin at 25 degrees C for a series of peptides having the general structure N-p-toluyl-(X)(n) (n =1-3), where X is glycine, alanine, or sarcosine. Apparent residue group contributions were calculated from permeability coefficients, P(RX), using the equation Delta(Delta G degrees )(X) = -RT ln(P(RX)/P(RH)). Multiple linear least-squares regression analysis performed for the set of 14 permeants yielded the best correlation (r(2) = 0.9993) when the following permeant descriptors were utilized: side-chain nonpolar surface area, number of -CONH- residues, number of toluyl-CON(Me)- residues, and number of other -CON(Me)- residues. The backbone -CONH- residue contribution in peptides, 4.6 kcal/mol, is significantly lower than that obtained for a single isolated -CONH- (>6 kcal/mol), suggesting a possible influence of intramolecular hydrogen bonding. Under closer scrutiny, Delta(Delta G degrees )(X) for the Ala and Gly residues decrease with increasing peptide length. The effect of N-methylation is also highly dependent on position and number of N-methyl groups on the molecule (Delta(Delta G degrees )(X) = -0.5 to -2.2 kcal/mol). These nonadditivities may be rationalized by considering the effects of peptide length and N-methylation on membrane-induced intramolecular hydrogen bonding leading to various folded conformations.

We report here the isolation of a novel enzyme from bovine neurointermediate pituitary which catalyzes the conversion of alpha-hydroxybenzoylglycine to benzamide. This enzyme, termed HGAD (alpha-hydroxyglycine amidating dealkylase), is a soluble protein with an apparent molecular mass of 45 kDa and no apparent cofactor requirement. Addition of HGAD to purified neurointermediate pituitary PAM (peptidylglycine alpha-amidating monooxygenase, EC 1.14.17.3) increases the rate of formation of amide products by an order of magnitude. Sequential additions of PAM and HGAD gave results consistent with PAM first catalyzing the formation of an intermediate that is subsequently, in a separate reaction, converted by HGAD to the final amide product. Experiments with olefinic inactivators demonstrate that HGAD is not required for turnover-dependent inactivation of PAM and, correspondingly, that HGAD activity is not affected by inactivators of PAM. As expected, HGAD has no effect on the rate of PAM-catalyzed sulfoxidation, where a reaction analogous to that occurring during amidation of glycine-extended substrates is not possible. On the basis of these results, we propose that peptide C-terminal amidation in neurointermediate pituitary is a two-step process, with PAM first catalyzing the conversion of a glycine-extended peptide to the alpha-hydroxyglycine derivative, which is in turn converted to the final amide product by HGAD.

Peptidylglycine alpha-amidating monooxygenase (PAM, EC 1.14.17.3) catalyzes the formation of alpha-amidated peptides from their glycine-extended precursors, thus playing a key role in the processing of peptide neurohormones. We now report that PAM readily catalyzes three alternate monooxygenase reactions--sulfoxidation, amine N-dealkylation, and O-dealkylation. Thus, (4-nitrobenzyl)thioacetic acid is converted to the analogous sulfoxide, N-(4-nitrobenzyl)glycine is converted to 4-nitrobenzylamine and glyoxylate, and [(4-nitrobenzyl)oxy]acetic acid is converted to 4-nitrobenzyl alcohol and glyoxylate. All these new activities display the characteristics expected for the normal PAM-catalyzed reductive oxygenation pathway and produce an equimolar amount of glyoxylate together with the heteroatom-containing dealkylation products. The ester [(4-methoxybenzoyl)oxy]acetic acid is not a PAM substrate, but is instead a good competitive inhibitor (KI = 0.48 mM). In addition, we report that the olefinic substrate analogues trans-benzoylacrylic acid and 4-phenyl-3-butenoic acid are potent time-dependent inactivators of PAM, with inactivation exhibiting the characteristics expected for mechanism-based inhibition. Monoethyl fumarate is also a time-dependent inactivator of PAM. Finally, we introduce several small non-peptide substrates for PAM by demonstrating that PAM catalyzes the transformation of hippuric acid and several ring-substituted derivatives to the corresponding benzamides and glyoxylic acid, with the most facile substrate of this class being 4-nitrohippuric acid. These compounds are the smallest amide substrates yet reported for PAM, and it is thus apparent that only the minimal structure of an acylglycine is required for PAM-catalyzed oxygenative amidation.

The bifunctional peptidylglycine alpha-amidating enzyme catalyzes the C-terminal amidation of glycine-extended peptides. The first enzyme activity, peptidylglycine alpha-hydroxylating monooxygenase, catalyzes the oxygen-, ascorbate-, and copper-dependent formation of alpha-hydroxyglycine derivatives. These are substrates for the second enzyme activity, peptidylamidoglycolate lyase, which catalyzes their breakdown to the corresponding C-terminal amidated peptide and glyoxylate as final products. Kinetic and isotope effect studies were carried out with N-benzoylglycine as a substrate at pH 6.0 using monofunctional and bifunctional monooxygenase activities. Kinetic data indicate an equilibrium ordered mechanism, with hippuric acid binding first followed by oxygen. A potentially important difference between the two monooxygenase activities is that product release occurs more slowly from the bifunctional enzyme, indicating an influence of the lyase domain on release of alpha-hydroxyglycine product to solution. Intrinsic isotope effects for the C-H bond cleavage were measured for the monofunctional form of the enzyme using a double-label tracer method, yielding 10.6 +/- 0.8 and 1.20 +/- 0.03 for the primary and alpha-secondary deuterium intrinsic isotope effects, respectively. These values are identical to previous measurements for the analogous enzyme system, dopamine beta-monooxygenase [Miller, S. M., and Klinman, J. P. (1985) Biochemistry 24, 2114-2127]. The identity of intrinsic isotope effects for peptidylglycine alpha-hydroxylating monooxygenase and dopamine beta-monooxygenase with substrates of comparable reactivity (N-benzoylglycine and dopamine, respectively) extends similarities between the two enzymes significantly beyond sequence homology and cofactor requirements.

The spontaneous and enzyme-catalyzed hydrolyses of a number of saturated oxazolinones have been investigated by following the decrease in ultraviolet absorbance which occurs on ring opening. Rate constants determined for the hydrolyses of saturated oxazolinones indicate the high reactivity of the oxazolinone carbonyl carbon toward nucleophilic attack. Oxazolinones have been shown to be good substrates for a number of hydrolytic enzymes. 2-Phenyloxazolin-5-one and 4,4-dimethyl-2-phenyloxazolin-5-one react rapidly with α-chymotrypsin, trypsin, and papain, forming relatively stable acyl-enzymes. The acylation reaction may be observed directly, providing evidence for the three-step mechanism of hydrolysis and a method for titration of the enzyme active sites. 2-Phenyloxazolin-5-one and p-nitrophenyl hippurate have been compared as substrates for α-chymotrypsin and ox liver carboxylesterase. Kinetic data obtained for the achymotrypsin-catalyzed hydrolysis of DL-4-(p-hydroxybenzyl)-2-phenyloxazolin-5-one provide an estimate of the optical specificity shown in both acylation and deacylation reactions.

Kinetic evidence has been obtained which is consistent with the formation of an acyl-enzyme intermediate in the hydrolysis of butyrate esters catalyzed by pig liver carboxylesterase. A study of the pig liver carboxylesterase- catalyzed hydrolysis of phenyl butyrate revealed activation by substrate and by modifiers such as benzene. This activation has been interpreted in terms of a classical kinetic scheme involving sites for substrate and modifier on a single enzyme molecule. For a series of activated esters, the catalytic rate constants for the carboxylesterase-catalyzed hydrolyses were found to be relatively insensitive to changes in the acyl group, indicating that binding is not responsible for the high reactivity of these systems. Kinetic and other data suggest that there are possibly some different elements of essential chemistry between the esterases and the proteinases.

The pH dependence (pH 4.5-10.5) of the hydrolysis of seven hippuric acid esters (C6H5CONHCH2C-O2CR1R2CO2H: 1a: R1 = R2 = H; 1b: R1 = R2 = CH3; 1c: R1 = H, R2 = p-ClC6H4; 1d: R1 = H, R2 = C2H5; 1e: R1 = H, R2 = (CH3)2CHCH2; 1f: R1 = H, R2 = C6H5; 1g: R1 = H, R2 = C6H5CH2) by bovine carboxypeptidase A has been investigated, and the pH dependence of the substrate activation of 1a-c and the substrate inhibition of 1d-g have been compared. For all seven esters the catalytically productive binding of the first substrate molecule depends on enzymatic pKa values of 6.0 and 9.1. For 1d, 1e, and 1g the rate of hydrolysis (k2app) of this complex is pH independent, whereas for 1f k2app depends on a pKa of 5.9. The rate of hydrolysis (k3app) of the 1:2 enzyme-substrate complex (ES2) is pH independent for 1d-g, but for 1a-c k3app depends on the following pKa values: 1a, 6.1 and 9.1; 1b, 5.4; 1c, 6.6. The pH dependences of k2app for 1f and k3app for 1c are rationalized by the presence of catalytically nonproductive species. Equivalent ES2 species are believed to be productive for 1c-g; however, the productive ES2 species for 1b must be quite different.