Bacillus subtilis aminopeptidase: Specificity toward amino acid amides,dipeptides, and oligopeptides

Bacillus subtilis aminopeptidase hydrolyzed amino acid amides with a specificity similar to that determined using amino acyl-β-naphthylamides, but at much greater catalytic rates. Neutral and basic amino acid amides were the best substrates. A series of Leu and Lys NH₂-terminal dipeptides hydrolyzed by Co²⁺-activated aminopeptidase showed that the $\text{k}{}_{\mathrm{<mtext>cat</mtext>}}\text{K}{}_{\mathrm{m}}$ ratios for the Lys substrates were fourfold greater than the corresponding Leu substrates and that catalytic differences reflected the identity of COOH terminal residues. Greatest catalytic rates were obtained when aromatic residues were in the COOH terminal position of the substrate (Trp, Tyr, Phe); but, significant hydrolysis was achieved when aliphatic residues were COOH-terminal in the dipeptide. The Co²⁺-activated enzyme would not hydrolyze peptide bonds composed of the imide nitrogen of Pro, thus, bradykinin was not a substrate. However, the Co²⁺-activated enzyme removed sequentially the first four residues from eledoisin-related peptide and the A chain of bovine insulin.     

The pH dependence of peptide hydrolysis by nitrocarboxypeptidase A

Hydrolysis of benzyloxycarbonyl-GlyGlyPhe by nitro(Tyr 248)carboxypeptidase A over the pH range 4.88–8.04 has been examined. The nitroenzyme retains appreciable activity near pH 6.5, and the limiting value of K_m is scarcely affected. The peptidase activity has a pH dependence characterized by the following parameters: pK_E1 of 6.37 ± 0.19 and pK_E2 of 6.60 ± 0.17 in $\text{k}{}_{\mathrm{<mtext>cat</mtext>}}\text{K}{}_{\mathrm{m}}$, and apparent pK of 5.59 ± 0.06 in K_cat. A spectroscopic pK of 6.75 ± 0.01, attributable to the nitro-Tyr 248 residue, has been determined. This correlates with the base-limb pK_E2 in the $\text{k}{}_{\mathrm{<mtext>cat</mtext>}}\text{K}{}_{\mathrm{m}}$ profile, which appears to be shifted from a higher value, pK_E2 of 9.0, for the native enzyme. The single (acid-limb) pK which characterizes the k_cat profile of the native enzyme is also found to be perturbed to a lesser extent by nitration. A kinetically competent reverse protonation mechanism, based on chemical modification and crystallographic evidence for the enzyme, is described.     

Dependence on pH of the activity of pig liver esterase

The dependence on pH of the kinetic parameters for the hydrolysis of phenyl acetate catalyzed by pig liver carboxylesterase was examined for purified high-isoelectric point and low-isoelectric point fractions of enzyme that were separated by isoelectric focusing. The values of k_cat are half-maximal at pH 4.3 and 5.1 for the high- and low-isoelectric point forms, respectively, and show a shallow dependence on pH with a value of n = 0.5. The absence of a change in the pH dependence of k_cat for the high-isoelectric point enzyme in the presence of high concentrations of methanol, which reacts with the acetyl-enzyme intermediate to give methyl acetate, provides evidence that the pH dependence is not caused by a change in rate-determining step. This means that if an imidazole group is involved in catalysis its pK must be perturbed downward by 2–3 units. The pH dependence of $\text{k}{}_{\mathrm{<mtext>cat</mtext>}}\text{K}{}_{\mathrm{m}}$ is biphasic with apparent pK values for dissociations of the free enzyme near 7 and 4 for both the high- and low-isoelectric point enzymes. Inhibition by a second molecule of substrate and by methanol are strongest for high-pH forms of the enzyme.     

Base-group specificity at the primary recognition site of ribonuclease T1 for minimal RNA substrates

Kinetic studies on the RNase T₁-catalyzed transesterification of 12 dinucleoside monophosphates, N_p¹N² (N¹ = A, C, and U; N² = A, C, G, and U) at pH 5, 25 °C, and 0.2 m ionic strength, revealed that the catalytic efficiency ($\text{k}{}_{\mathrm{<mtext>cat</mtext>}}\text{K}{}_{\mathrm{m}}$) for GpN substrates (H. L. Osterman, and F. G. Walz, Jr., 1978, Biochemistry, 17, 4142) was ~10⁶-fold greater than corresponding ApNs and at least 10⁸-fold greater than corresponding CpNs and UpNs. The catalytic activity with ApN substrates survives phenol extraction which indicates (along with other criteria) that it is intrinsic to RNase T₁ and is not due to trace contamination by other nucleases. Circumstantial evidence is presented which suggests that homologous GpN and ApN substrates bind productively at different sites on the enzyme. The results of steady-state kinetic studies of RNase T₁ with IpNs (N = C and U) were compared with those for GpNs and indicated that the primary effect of the guanine 2-NH₂ group is to enhance substrate binding at the primary recognition site by ~2.6 kcal/mol. Values of ($\text{k}{}_{\mathrm{<mtext>cat</mtext>}}\text{K}{}_{\mathrm{m}}$) showed the order NpC > NpU (N = A, G, and I) which evidences the existence of a subsite for the leaving nucleoside group that prefers cytidine: interactions at this subsite are reflected in k_cat rather than K_m.     

Mechanism of action of thrombin on fibrinogen. The reaction of thrombin with fibrinogen-like peptides containing 11, 14, and 16 residues

The following peptides were synthesized by classical methods in solution: Ac-Gly-Gly- Val-Arg-Gly-Pro-Arg-Val-Val-Glu-Arg-NHCH₃ (A), Ac-Ala-Glu-Gly-Gly-Gly-Val- Arg-Gly-Pro-Arg-Val-Val-Glu-Arg-NHCH₃ (B), and Ac-Phe-Leu-Ala-Glu-Gly-Gly- Gly-Val-Arg-Gly-Pro-Arg-Val-Val-Glu-Arg-NHCH₃ (C). The rates of hydrolysis of the Arg-Gly bond of these three peptides by thrombin were measured, and the values of $\text{k}{}_{\mathrm{<mtext>cat</mtext>}}\text{K}{}_{\mathrm{m}}$ were found to be 0.05 × 10^?7 (A), 0.02 × 10^?7 (B), and 1.6 × 10^?7 (C) [(NIH units/ liter)s]^?1. The value of$\text{k}{}_{\mathrm{<mtext>cat</mtext>}}\text{K}{}_{\mathrm{m}}$ for peptide C is less than 1% of that for fibrinogen [although the value of k_cat itself, for peptide C (but not for A or B), is comparable to that for fibrinogen]. These results indicate that phenylanine and leucine at positions P₉ and P₈, respectively, play a key role in the reaction of thrombin with fibrinogen. The data also show that factors outside of the 16 residues of peptide C are important in determining the rate of hydrolysis of fibrogen by thrombin.     

Kinetics of H2O2-driven catalysis by a lytic polysaccharide monooxygenase from the fungus Trichoderma reesei

Owing to their ability to break glycosidic bonds in recalcitrant crystalline polysaccharides such as cellulose, the catalysis effected by lytic polysaccharide monooxygenases (LPMOs) is of major interest. Kinetics of these reductant-dependent, monocopper enzymes is complicated by the insoluble nature of the cellulose substrate and parallel, enzyme-dependent, and enzyme-independent side reactions between the reductant and oxygen-containing cosubstrates. Here, we provide kinetic characterization of cellulose peroxygenase (oxidative cleavage of glycosidic bonds in cellulose) and reductant peroxidase (oxidation of the reductant) activities of the LPMO TrAA9A of the cellulose-degrading model fungus Trichoderma reesei. The catalytic efficiency (kcat/Km(H2O2)) of the cellulose peroxygenase reaction (k_cat = 8.5 s⁻¹, and Km(H2O2)=30μM) was an order of magnitude higher than that of the reductant (ascorbic acid) peroxidase reaction. The turnover of H₂O₂ in the ascorbic acid peroxidase reaction followed the ping-pong mechanism and led to irreversible inactivation of the enzyme with a probability of 0.0072. Using theoretical analysis, we suggest a relationship between the half-life of LPMO, the values of kinetic parameters, and the concentrations of the reactants.     

On the mechanism of the reaction of tris(hydroxymethyl)aminomethane with activated carbonyl compounds: A model for the serine proteinases

The pH dependence of the reaction of tris(hydroxymethyl)aminomethane (Tris) with the activated carbonyl compound 4-trans-benzylidene-2-phenyloxazolin-5-one (I) is given by the equation $\text{k′}{}_2\text{=}\text{k}{}_{\mathrm{<mtext>b</mtext>}}\text{K}{}_{\mathrm{<mtext>a</mtext>}}\text{(K}{}_{\mathrm{<mtext>a</mtext>}}\text{+ [}\text{H}{}^{\mathrm{+}}\text{])}\text{+}\text{k}{}_{\mathrm{<mtext>a</mtext>}}\text{[}\text{OH}{}^{\mathrm{?}}\text{]K}{}_{\mathrm{<mtext>a</mtext>}}\text{(K}{}_{\mathrm{<mtext>a</mtext>}}\text{+ [}\text{H}{}^{\mathrm{+}}\text{])}$, where K_a is the dissociation constant of TrisH⁺. Spectrophotometric experiments show that the Tris ester of α-benzamido-trans-cinnamic acid is formed quantitatively over a range of pH values, regardless of the relative contribution of k_b and k_a terms to k′₂. Hence, both terms refer to alcoholysis. While the mechanism of the reaction is not determined unequivocally in the present work, the magnitude of the k_b term, together with its dependence on the basic form of Tris, suggests that ester formation is occurring by nucleophilic attack of a Tris hydroxyl group on the carbonyl carbon of the oxazolinone, with intramolecular catalysis by the Tris amino group. The rate enhancement due to this group is at least 10² and possibly of the order 10⁶. This system is compared with other model systems for the acylation step of catalysis by serine esterases and proteinases.     

Protein affinities for small molecules: conceptions and misconceptions.

There is much confusion and error in published treatments of data for multiple binding of ligands (e.g., substrates) by proteins (e.g., enzymes). There is a widespread impression that if the equilibrium binding, r, of ligand, A, by a protein with n sites can be fitted to an equation with two hyperbolic terms, i.e., $\text{r=}\text{n}{}_{\mathrm{\alpha }}\text{k}{}_{\mathrm{\alpha }}\text{(A)}\text{1+k}{}_{\mathrm{\alpha }}\text{(A)}\text{+}\text{n}{}_{\mathrm{\beta }}\text{k}{}_{\mathrm{\beta }}\text{(A)}\text{1+k}{}_{\mathrm{\beta }}\text{(A)}\text{(n}{}_{\mathrm{\alpha }}\text{+n}{}_{\mathrm{\beta }}\text{=n)}$ then k_β and k_β are always the intrinsic binding constants for two sets of sites. Such a conclusion is often incorrect. For example, in many cases, the protein is constituted of identical protomers with initially identical sites for binding ligands, and yet graphical representations of the binding data appear to behave as if the sites are partitioned between two classes. Although the use of a linear combination of hyperbolic terms to represent binding of ligands by macromolecules always yields empirical parameters k_α, k_β … k_λ, they cannot correspond to site binding constants when there are interactions between sites. In some circumstances their values may even be imaginary, complex numbers. On the other hand, stoichiometric binding constants can be assigned unambiguously without making any assumption regarding the nature of the interactions among binding sites. These principles are illustrated concretely by analyses of binding measurements for several different proteins containing two to six sites.     

Structure-activity relationships in papain and bromelain ligand interactions

The parameters K_m and k_cat were determined for 16 methyl hippurates (CH₃OCOCH₂NHCOC₆H₄-X) hydrolyzed by papain. A simple linear relationship is found between log $\text{1}\text{K}{}_{\mathrm{m}}$ and the hydrophobic substituent constant π. It is found that log k_cat is parabolically related to π. The results with papain are compared with results obtained by Hawkins and Williams with the enzyme bromelain. The two enzymes behave in a similar fashion.     

Presteady-state kinetic study of the elementary processes in the chymotrypsin-catalyzed hydrolysis of specific ester substrate. Rate-limiting association process due to the secondary binding.

Presteady-state kinetic studies of α-chymotrypsin-catalyzed hydrolysis of a specific chromophoric substrate, N-(2-furyl)acryloyl-l-tryptophan methyl ester, were performed by using a stopped-flow apparatus both under [E]₀ ? [S]₀ and [S]₀ ? [E]₀ conditions in the pH range of 5–9, at 25 °C. The results were accounted for in terms of the three-step mechanism involving enzyme-substrate complex (E · S) and acylated enzyme (ES′); no other intermediate was observed. This substrate was shown to react very efficiently, i.e., the maximum of the second-order acylation rate constant ($\text{k}{}_2\text{K}{}_{\mathrm{s}}\text{′}\text{)}{}_{\mathrm{<mtext>max</mtext>}}\text{= 4.2 × 10}{}^7\text{M}{}^{\mathrm{?1}}\text{s}{}^{\mathrm{?1}}$. The limiting values of K_s′ (dissociation constant of E · S), K₂ (acylation rate) and k₃ (deacylation rate) were obtained from the pH profiles of these parameters to be 0.6 ± 0.2 × 10^?5 m, 360 ± 15 s^?1 and 29.3 ± 0.8 s^?1, respectively. Likewise small values were observed for K_i of N-(2-furyl)-acryloyl-l-tryptophan and N-(2-furyl)acryloyl-d-tryptophan methyl ester and K_m of N-(2-furyl)acryloyl-l-tryptophan amide. The strong affinities observed may be due to intense interaction of β-(2-furyl)acryloyl group with a secondary binding site of the enzyme. This interaction led to a $\text{k}{}_{\mathrm{?1}}\text{k}{}_2$ value lower than unity, i.e., the rate-limiting process of the acylation was the association, even with the relatively low k₂ value of this methyl ester substrate, compared to those proposed for labile p-nitrophenyl esters.     

Alpha-chymotrypsin: effects of bicyclic substrate geometry and hydrophobicity on reactivity

The α-chymotrypsin-catalyzed hydrolysis rates of p-nitrophenyl cyclopentane-carboxylate (I), p-nitrophenyl indan-2-carboxylate (II), and p-nitrophenyl spiro-[4.4]nonane-2-carboxylate (III) were measured at pH 8.1 in 20% methanol. After correction for variations in reactivity owing to stereoelectronic effects inherent to the substrates, the deacylation rate constants (k_c)_n of I and II are not significantly different. In $\text{(}\text{k}{}_{\mathrm{c}}\text{K}{}_{\mathrm{m}}\text{)}{}_{\mathrm{n}}$ II is 50 times more reactive than I, which demonstrates that the aromatic ring of the former substrate contributes significantly to its reactivity. The nearly equal reactivities of II and III indicate that the enzyme is rather insensitive to the geometry of the nonester-bearing ring of these compounds.     

Hydrolysis of a thiopeptide by cadmium carboxypeptidase A

Substitution of the active site zinc ion of carboxypeptidase A by cadmium yields an enzyme inactive towards ordinary peptide substrates. However, a substrate analog (BzGlyNHCH₂CSPheOH) containing a thioamide linkage at the scissile position is cleaved to the thioacid. The kinetic parameters and their pH dependencies are $\text{k}{}_{\mathrm{cat}}\text{K}{}_{\mathrm{m}}\text{= 5.04 × 10}{}^4\text{min}{}^{\mathrm{?1}}\text{M}{}^{\mathrm{?1}}$, decreasing with either acid or base (PK_E1 = 5.64, pK_E2 = 9.55), and k_cat = 1.02 × 10² min^?1, decreasing with acid (pK_ES = 6.61). The thiopeptide is less efficiently cleaved by native (zinc) carboxypeptidase A. This cadmium-sulfur synergism supports a mechanism wherein the substrate amide is activated by metal ion coordination to its (thio) carbonyl.     

6-Phosphogluconolactonase from Zymomonas mobilis: An enzyme of high catalytic efficiency

The enzyme 6-phosphogluconolactonase (EC 3.1.1.31) is present at high levels in Zymomonas mobilis cells. A simple procedure for its isolation involving dye-ligand chromatography and gel filtration has resulted in a 500-fold purification with high recovery. The purified enzyme is a monomer of 26 kDa, and has a high catalytic efficiency with $\text{k}{}_{\mathrm{cat}}\text{K}{}_{\mathrm{m}}$ of 9 × 10⁷ M⁻¹ s⁻¹ at 25° C. Two assay procedures for the enzyme are compared, and a simple method of obtaining a solution of 6-phosphoglucono-δ-lactone relatively free of other metabolites is presented.     

Opposite modulation by uncoupling and electron transport limitation of the Kmapp of ADP for photophosphorylation

The $\text{K}{}_{\mathrm{m}}{}^{\mathrm{(app)}}$ of ADP for photophosphorylation in lettuce chloroplasts was measured both at various light intensities and in the presence of various uncoupler (nigericin + K⁺) concentrations. Lowering the light intensity results in both, a decrease in the rate of phosphorylation and a several fold $\text{decrease}$ in the $\text{K}{}_{\mathrm{m}}{}^{\mathrm{(app)}}$ of ADP for the reaction. However, when increasing concentrations of the uncoupler nigericin + K⁺ are employed, the rate of photophosphorylation is decreased but a several-fold $\text{increase}$ in the $\text{K}{}_{\mathrm{m}}{}^{\mathrm{(app)}}$ of ADP for the reaction is observed. The results are discussed in terms of the chemiosmotic hypothesis. It is suggested that these effects might indicate the existence of a mechanism controlling the rate of ATP formation which is different than the formation of the electrochemical gradient.     

The explicit analysis of consecutive pseudo-first-order reactions: Application to kinetics of thiolysis of dithiasuccinoyl (Dts) amino acids

An explicit set of general methods for the experimental determination of the rates k₁ and k₂ of consecutive pseudo-first-order reactions is described and discussed. These rely on the direct simultaneous analytical quantitation of the starting material, intermediate, and product of the reaction, and thus differ from present techniques based on measurement of coreactant consumption or coproduct appearance. The quantity $\text{k}{}^{\mathrm{<mtext>env</mtext>}}\text{=}\text{k}{}_1\text{k}{}_2\text{(k}{}_1\text{+ k}{}_2\text{)}$ is shown to define a good “envelope” approximation to product formation according to the simple law 100% [1 ? exp(?k^envt)]. The theory of envelopes is useful for comparing overall rates of reactions with widely differing values of $\text{κ =}\text{k}{}_2\text{k}{}_1$. The kinetic pattern of thiolysis of dithiasuccinoyl amino acids to carbamoyl disulfide intermediates to product free amino acids is analyzed and shown to agree quantitatively with theory.     

Kinetic studies on the reaction of ethylenediaminetetraacetatochromium(III) complex with hydrogen peroxide

The rate of reaction of [Cr(III)Y]_aq (Y is EDTA anion) with hydrogen peroxide was studied in aqueous nitrate media [μ = 0.10 M (KNO₃)] at various temperatures. The general rate equation, Rate = $\text{k}{}_1\text{+}\text{k}{}_2\text{K}{}_1\text{[H}{}^{\mathrm{+}}\text{]}{}^{\mathrm{?1}}\text{1 +}\text{K}{}_1\text{[H}{}^{\mathrm{+}}\text{]}{}^{\mathrm{?1}}$ [Cr(III)Y]_aq[H₂O₂] holds over the pH range 5–9. The decomposition reaction of H₂O₂ is believed to proceed via two pathways where both the aquo and hydroxo-quinquedentate EDTA complexes are acting as the catalyst centres. Substitution-controlled mechanisms are suggested and the values of the second-order rate constants k₁ and k₂ were found to be 1.75 × 10^?2 M^?1 s^?1 and 0.174 M^?1 s^?1 at 303 K respectively, where k₂ is the rate constant for the aquo species and k₂ is that for the hydroxo complex. The respective activation enthalpies (ΔH^*₁ = 58.9 and ΔH^*₂ = 66.5 KJ mol^?1) and activation entropies (ΔS^*₁ = ?85 and ΔS^*₂ = ?40 J mol^?1 deg^?1) were calculated from a least-squares fit to the Eyring plot. The ionisation constant pK₁, was inferred from the kinetic data at 303 K to be 7.22. Beyond pH 9, the reaction is markedly retarded and ceases completely at pH ? 11. This inhibition was attributed in part to the continuous loss of the catalyst as a result of the simultaneous oxidation of Cr(III) to Cr(VI).