Practical acid dissociation constants,temperature coefficients,and buffer capacities for some biological buffers in solutions containing dimethyl sulfoxide between 25 and −12 °C

Acid dissociation constants (K_a), usually expressed as pK_a (-logK_a) can be considered as indices of acid-base equilibria in solution and their evaluation under the solution conditions that exist during the exposure of biological systems to low temperatures are as important as the measurement of pH per se. The assignment of pH1 standards to define pH1 scales in the binary mixed solvent, dimethyl sulfoxide-water (27), has provided the basis for measuring the pK_a1 values of some biological buffers in mixtures of Me₂SO and H₂O which have particular relevance to studies which demonstrate the “pH-dependent” recovery of smooth muscle after low-temperature storage (9, 31). “Practical” ionization conslants in water (pK′_a) and in 20% ($\text{w}\text{w}$) and 30% ($\text{w}\text{w}$) dimethylsulfoxide-water (pK_a1) have been measured by potentiometric titration of a range of zwitterionic buffer compounds at 25, 0, ?5.5, and ?12 °C together with the respective buffer capacities and temperature coefficients. Measurements have been made with reference to the relevant standard states for each solvent system, thereby endowing the values with as much thermodynamic significance as possible.     

The self-association of chorismate mutase/prephenate dehydratase from Escherichia coli K12

The state of association of chorismate mutase/prephenate dehydratase (EC 5.4.99.5/ 4.2.1.51) from E. coli K12 has been studied using ultracentrifugal techniques. The smallest species inferred is a dimer of molecular weight 73,000–84,000, with a $\text{s}{}_{\mathrm{<mtext>20,w</mtext>}}{}^0$ of 5.02 S at pH 8.2, I = 0.013 M. This species undergoes a concentration-dependent self-association which results in an equilibrium mixture of dimer, tetramer, and probably octamer, with a M_r of 164,000 at an enzyme concentration of 8.0 mg/ml under the same conditions. Addition of the feedback inhibitor phenylalanine (2 mm) or increase in ionic strength (I = 0.40 M), or a decrease in pH to 7.4 displaces this equilibrium toward the higher-molecular-weight forms of the enzyme, resulting in M_r values of 273,000, 254,000, and 257,000, respectively. This behavior partially explains the allosteric kinetics and inhibitor binding observed previously with this enzyme.     

Purification and characterization of an intracellular N-terminal exopeptidase from Streptococcus durans

An intracellular N-terminal exopeptidase isolated from cell extracts of Streptococcus durans has been purified 470-fold to homogeneity (specific activity of 12.0 μmol/min per mg). In the absence of thiol compounds, the purified aminopeptidase undergoes a slow oxidation with a 70% loss of activity, which can be restored by the addition of 2 mM β-mercaptoethanol. The purified aminopeptidase (M_r 300 000) preferred L-peptide and arylamide substrates with small nonpolar or basic side chains. SDS electrophoresis yielded a single protein band corresponding to a molecular weight of 49 400, suggesting that the native enzyme is a hexameric protein. The enzyme-catalyzed hydrolysis of $\text{L}\text{-}\text{alanyl}\text{-p-}\text{nitroanilide}$ exhibited a bell-shaped pH dependence for log V_max/K_m(pK₁ = 6.35; pK₂ = 8.50) while the log V_max versus pH profile showed only an acid limb (pK = 6.35). Methylene blue-sensitized photooxidation of the enzyme resulted in the complete loss of activity, while L-leucine, a competitive inhibitor, partially protected against this inactivation. Amino acid analysis indicated that this photooxidative loss of activity corresponded to the modification of one histidine residue per enzyme monomer. N-Ethylmaleimide (100 mM) caused a 78% reduction in enzyme activity. Treatment of the enzyme with 1.0 mM hydrogen peroxide resulted in the oxidation of two cysteine residues per enzyme monomer and caused a 70% decrease in the catalytic activity.     

Hydrodynamic properties of rat hepatic prolactin receptors

The hydrodynamic properties of rat hepatic prolactin receptors have been determined by a combination of gel chromatography and ultracentrifugation. Prolactin receptors were detergent extracted from partially purified plasma membranes prepared from female rat livers. Fifteen different nonionic detergents were tested for solubilizing prolactin receptors, including Triton X-100, Polyoxyethylene W-1, Lubrol WX, detergents of the Tween and Brij series, and digitonin. When the receptors were detergent solubilized after ligand was bound to the receptor, 1% Triton X-100 had the highest efficacy of solubilization. However, if the receptors were solubilized prior to exposure to ligand, maximum binding was to receptors solubilized with 0.25% Triton X-100. The K_d of 43.2–74.5 pM for binding to the soluble receptor was three to fivefold lower than the K_d for the membrane receptor. Gel chromatography (Bio-Gel A-1.5m, 2.5 × 50 cm) of the soluble receptor indicated a Stokes radius (R_s) of 5.0 nm for the hormonereceptor-detergent complex. The hydrodynamic properties of the receptor-detergentligand complex were determined by centrifugation in 5–20% sucrose gradients in H₂O and in D₂O. They are $\text{v}\text{?}\text{= 0.7; s}{}_{\mathrm{<mtext>20,</mtext><mtext>w</mtext>}}\text{= 4.7;}\text{f}\text{f}{}_0\text{= 1.49; M}{}_{\mathrm{<mtext>r</mtext>}}\text{= 118,000}$ for the complex, 73,000 for the receptor alone. Approximately 0.22 mg of Triton X-100 is estimated bound per milligram of protein. This represents about 25 mol detergent/mol receptor.     

The effect of NADP on the subunit structure and activity of spinach chloroplast glyceraldehyde-3-phosphate dehydrogenase

Spinach chloroplast glyceraldehyde phosphate dehydrogenase (d-glyceraldehyde-3-phosphate: NADP oxidoreductase, phosphorylating; EC 1.2.1.13) is an equilibrium mixture of aggregates of a basic protomer (M_r about 145,000) and is active with both NADP and NAD. The enzyme is primarily “tetrameric” (M_r about 600,000), although minor amounts of smaller and larger oligomers are also found. Gel chromatography in buffer containing 30 μm NADP results in depolymerization of the enzyme, mainly to protomers. NAD does not dissociate and counteracts this effect of NADP.The apparent K_m values of the protomers are 7 μm (NADP) and 8 μm (NAD). The aggregates with a M_r > 10⁶ have properties similar to the protomers. The tetramer as first isolated has higher M_m values for NADP (380 μm) and NAD (48 μm), but its apparent affinity for NADP is further decreased by repeated gel filtrations in buffer or by a single one in buffer containing NAD. Such preparations display nonlinear kinetics when NADP is the varied substrate and have a K_m (NADP) of about 1.5–3.3 μm. All these effects are reversible.V values are apparently the same in all enzyme forms and the $\text{V (}\text{NADP}\text{)}\text{V (}\text{NAD}\text{)}$ ratio always approaches 2. Since, however, the enzyme is presumably dissociated by the NADP concentrations required for a “saturating” assay, the significance of V (NADP) seems questionable.     

Effects of 2,4-substituents of deuteropheme upon redox potentials of horseradish peroxidases.

The effects of pH on redox potentials of horseradish peroxidase-A and -(B + C) and of their heme-substituted enzymes with mesoheme, deuteroheme, chlorocruoroheme, and diacetyldeuteroheme were investigated. The slope in the plot of E_o against pK₃ (a measure of basicity of pyrrole nitrogen) was found to be close upon 59 $\text{mV}\text{p}\text{K}{}_3$ unit. It was also found that the ratio of ΔpK_r to ΔpK₃ was about 0.1 while that of ΔpK_o to ΔpK₃ was almost unity. Here, K_r and K_o stand for heme-linked proton dissociation constants in the ferrous and ferric peroxidases, respectively. The difference of either pK_r or pK_o between two isoenzyme preparations was about 1.6. These results support the previous conclusion (Arch. Biochem. Biophys. 165, 725, 1974 that K_r represents a proton dissociation constant of a distal amino acid residue and that there is a strong hydrogen bonding between its base and the water oxygen atom as a sixth ligand in the ferric state of peroxidases. The difference of redox potentials at pH 8.5 between two natural isoenzyme preparations, amounting to 88 mV, was attributed to the change in the hydrogen bonding strength caused by the difference in basicity of two distal amino acid residues. A possibility that approximate redox potentials of hemoproteins can be determined by analysis of several factors is discussed.     

Bovine fibrinogens: Reversible polymer formation

Reversible flbrinogen polymer formation was examined at pH 6.6 and Γ/2 0.3. The equilibrium fraction of fibrinogen present as polymer, (P_mf)_e, was determined by gel filtration for fibrinogen concentrations, F_O, from 48 to 166 μm. Using F_O in molarity, the experimental relation is ln [F_O(P_mf)_e] = 3.53 ln[F_O(1 ? (P_mf)_e)] + 23.73. This relation and attendant confidence limits are examined assuming, during filtration, that the original polymer population is either stable or selected polymer species dissociate to monomer. The possibility that all polymers are open is excluded since the calculated microscopic association constant would then increase with F_O. Acceptable models are based on the assumptions that polymers are open, with association constant K_a, until restricted by closure, with association constant K_r, at an integral degree of polymerization, n. Values are selected on the basis that interaction parameters are independent of F_O and that the required molar decrease in free energy is a minimum. Assuming polymer stability, the experimental relation at 273 °K gives $\text{n = 4,}\text{K}{}_{\mathrm{r}}\text{K}{}_{\mathrm{a}}\text{= 1.2 m,}\text{and}\text{K}{}_{\mathrm{a}}$ = 736 m^?1. Temperature dependence gives $\text{ΔH= ?16.9 kcal/mol}\text{and}\text{ΔS}{}^{\mathrm{O}}{}_{\mathrm{a}}$ = ?48.8 e.u. $\text{K}{}_{\mathrm{r}}\text{K}{}_{\mathrm{a}}$ indicates a relation between changes in entropy. The probability is >0.90 that $\text{K}{}_{\mathrm{r}}\text{K}{}_{\mathrm{a}}\text{? 56 m}$, which indicates a greater loss of degrees of freedom on closure than on association. Conclusions are not altered by the assumption that only the closed polymer species is stable. As ionic strength is decreased at pH 6.6, K_a increases. The clotting time of an otherwise constant system decreases as system P_mf is increased.     

Inactivation of phosphofructokinase by dialdehyde-ATP.

Rabbit muscle phosphofructokinase (PFK) is rapidly inactivated by a 2′,3′-dialdehyde derivative of adenosine triphosphate (dialdehyde-ATP). When allowed to react with 0.6 mm dialdehyde-ATP in 0.1 m borate buffer (pH 8.6) containing 0.2 mm EDTA and 0.5 mm dithiothreitol, PFK loses essentially all activity (99%) in 30 min. The modified PFK remains inactive following dialysis of the reaction mixture against sodium borate (pH 8.0) containing fructose diphosphate, EDTA, and dithiothreitol. Experiments with [¹⁴C]dialdehyde-ATP show that 99% inactivation of PFK corresponds to incorporation of 3 to 4 mol of the ATP analog per PFK protomer. The inactivation of PFK with dialdehyde reagent is not caused by dissociation of the 340,000 M_r, tetramer to the 170,000 M_r dimer, as determined by analytical ultracentrifugation. Adenosine diphosphate or ATP protect PFK from inactivation by dialdehyde-ATP at pH 8.6, but fructose 6-phosphate, cyclic 3′,5t$\text{-}\text{?}$adenosine monophosphate, or fructose diphosphate, which protect PFK from modification by pyridoxal phosphate, provide little protection from inactivation. Amino acid analyses of dialdehyde-inactivated PFK and of a control sample of the enzyme were compared following reaction of each with 2,4-dinitrofluorobenzene. The results show that three or four lysine residues per PFK protomer are modified by dialdehyde-ATP. Additional data indicate that these lysine residues react with dialdehyde-ATP to form dihydroxymorpholine-like adducts rather than Schiff bases.     

Pig liver fatty acid synthetase: purification and physicochemical properties.

This paper reports the first detailed study of the physicochemical properties of a fatty acid synthetase multienzyme complex from a mammalian liver. Fatty acid synthetase from pig liver was purified by a procedure including the following main steps: (i) preparation of a clarified supernatant solution (50,000 g), (ii) ammonium sulfate fractionation, (iii) DEAE-cellulose chromatography to separate 11 S catalase from the 13 S fatty acid synthetase, (iv) a preparative sucrose density gradient step to remove a 7 S impurity, and (v) a calcium phosphate gel step to remove an unusual yellow 16 S heme protein to yield a colorless preparation. The purified fatty acid synthetase was colorless and showed a single symmetrical peak in sucrose density gradient and conventional sedimentation velocity experiments. Fatty acid synthetase was very stable at 4 °C in the presence of 1 mm dithiothreitol and 25% sucrose. Extrapolation to zero protein concentration yielded values of S^o_20,w = 13.3 S and D^o_20,w = 2.60 × 10^?7cm²/s for the sedimentation and diffusion coefficients of the enzyme. Frictional coefficient values of 1.55 and 1.56 × 10^?7 cm, respectively, were calculated from the values for the sedimentation and diffusion coefficients. Based on these frictional coefficient values, the Stokes radius of the enzyme was calculated to be 82.4 Å. Sedimentation and diffusion coefficient data yielded a molecular weight value of $\text{M}{}_{\mathrm{<mtext>w</mtext>}}\text{(}\text{s}\text{D}\text{) = 478,000}$ and sedimentation equilibrium data yielded a value of M_w = 476,000. Preliminary intrinsic viscosity measurements at 20 °C gave a value of 7.3 ml/g, indicating that the enzyme is somewhat asymmetric. This is supported by the value of 1.58 calculated for the frictional ratio and by the fact that the values for the sedimentation and diffusion coefficients are both slightly lower than expected for a globular protein of molecular weight 478,000. The enzyme possesses about 90 SH groups per molecule, assuming a molecular weight of 478,000. The ultraviolet absorption spectrum of the enzyme shows a maximum at 280 nm and an unusual shoulder at 290 nm. The fluorescence spectrum of the enzyme is dominated by tryptophan fluorescence and, over the excitation range of 260–300 nm, there is a single emission maximum at 344 nm.     

Proanthocyanidin polymers of fodder legumes

The structure of the condensed tannins of the most common fodder legumes is described. The number- ($\text{M}$_n) and weight-average ($\text{M}$_w ) MW of the polymers have been determined and most legume tannins have an approximately normal distribution of MWs with $\text{M}$_n values of 2000–4000.     

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.     

pH kinetic studies of the N-demethylation of N,N-dimethylaniline catalyzed by chloroperoxidase

The effect of pH on the kinetic parameters for the chloroperoxidase-catalyzed N-demethylation of N,N-dimethylaniline supported by ethyl hydroperoxide was investigated from pH 3.0 to 7.0. Chloroperoxidase was found to be stable throughout the pH range studied. Initial rate conditions were determined throughout the pH range. The V_max for the demethylation reaction exhibited a pH optimum at approximately 4.5. The K_m for N,N-dimethylaniline increased with decreasing pH, while the K_m for ethyl hydroperoxide varied in a manner paralleling V_max. Comparison of the $\text{V}{}_{\mathrm{<mtext>max</mtext>}}\text{K}{}_{\mathrm{m}}$ values for N,N-dimethylaniline and ethyl hydroperoxide indicated that the interaction of N,N-dimethylaniline with chloroperoxidase compound I was rate-limiting below pH 4.5, while compound I formation was rate-limiting above pH 4.5. The log of the $\text{V}{}_{\mathrm{<mtext>max</mtext>}}\text{K}{}_{\mathrm{m}}$ for ethyl hydroperoxide was independent of pH, indicating that chloroperoxidase compound I formation is not affected by ionizations in this pH range. The plot of the log of the $\text{V}{}_{\mathrm{<mtext>max</mtext>}}\text{K}{}_{\mathrm{m}}$ for N,N-dimethylaniline versus pH indicated an ionization on compound I with a pK of approximately 6.8. The plot of the log of the V_max versus pH indicated an ionization on the compound I-N,N-dimethylaniline complex, with a pK of approximately 3.1. The results show that chloroperoxidase can demethylate both the protonated and neutral forms of N,N-dimethylaniline (pK approximately 5.0), suggesting that hydrophobic binding of the arylamine substrate is more important in catalysis than ionic bonding of the amine moiety. For optimal catalysis, a residue in the chloroperoxidase compound I-N,N-dimethylaniline complex with a pK of approximately 3.1 must be deprotonated, while a residue in compound I with a pK of approximately 6.8 must be protonated.     

Purification and properties of Escherichia coli coenzyme A-transferase

The acetyl-CoA:acetoacetate-CoA-transferase has been purified 36-fold to homogeneity from an acetoacetate degradation operon (ato) constitutive mutant of Escherichia coli. The enzyme has the following physical properties: Stokes radius, 40.5 Å; diffusion coefficient (D₂₀,w), 5.32 × 10^?7 cm s^?1; sedimentation coefficient (s₂₀,w), 5.38S; molecular weight, 97,000 and a frictional ratio ($\text{f}\text{f}{}_0$) of 1.35. The enzyme is composed of two α subunits (M_r = 26,000) and two β subunits (M_r = 23,000). E. coli CoA-transferase contains six cysteine residues per mole of enzyme and no disulfide bonds. The native transferase reacts with 4 mol of p-chloromercuribenzoate per 97,000 g of enzyme. Two cysteine residues react rapidly with p-chloromercuribenzoate resulting in an 85% inactivation of enzyme activity. The reactivity of these two residues is enhanced at least fivefold in the presence of acetyl-CoA. Acetoacetate has no effect on the rate of reaction of p-chloromercuribenzoate with the enzyme. E. coli CoA-transferase is partially inactivated by acyl-CoA substrates in the absence of carboxylic acid substrates, presumably as the result of a metal-catalyzed acylation of the ?-amino group of a lysine residue near the active site. The enzyme utilizes a variety of short chain acyl-CoA and carboxylic acid substrates but exhibits maximal activity with normal and 3-keto substrates.     

Crystallizations and preliminary X-ray studies of calotropins DI and DII

Crystals of calotropin DI (M_r 23,400), have been prepared by microdialysis against 5% (w/v) polyethylene glycol 20,000 in water, pH 7.0. They have orthorhombic space group P2₁2₁2₁ with cell parameters $\text{a = 57.5}\text{A}\text{?}\text{, b = 86.2}\text{A}\text{?}\text{, c = 40.3}\text{A}\text{?}$. Crystals of calotropin DII (M_r 24,000), prepared by the same technique against 5% (w/v) polyethylene glycol 20,000 in phosphate buffer of low ionic strength, pH 7.0, display monoclinic space group C2 with cell parameters $\text{a = 135.8}\text{A}\text{?}\text{, b = 32.0}\text{A}\text{?}\text{, c = 47.7}\text{A}\text{?}\text{, β = 103.80 °}$. In both cases, there is only one molecule in the asymmetric unit.     

Crystallographic data for the DD-carboxypeptidase-endopeptidase of low penicillin sensitivity excreted by Streptomyces albus g.

The dd-carboxypeptidase-endopeptidase of low penicillin sensitivity that is excreted by Streptomyces albus G has been crystallized from a polyethylene glycol (M_r 6000 to 7500) solution at pH 8.0. X-ray examination of the prismatic crystals shows that the space group is P2₁ with unit cell dimensions $\text{a = 51.1}\text{A}\text{?}\text{, b = 49.7}\text{A}\text{?}\text{, c = 38.7}\text{A}\text{?}\text{, β = 100.6 °}$ and one molecule in the asymmetric unit. A crystal suspension made in 50 mm-Tris · HCl buffer (pH 8.0) supplemented with 5 mm-MgCl₂ and 16% ($\text{w}\text{v}$) polyethylene glycol exhibits enzyme activity on the substrate Ac₂-l-Lys-d-Ala-d-Ala.     

Sedimentation coefficients of self-associating species: I. Basic theory

Under the same solution conditions, the apparent weight average sedimentation coefficient, s_wa, and some quantities obtained from it can be combined with the equilibrium constant or constants, K_i, and the monomer concentration, c_I, obtained from sedimentation equilibrium, light scattering or osmotic pressure experiments on the same self-associating solute, so that the individual sedimentation coefficients, s_i, of the self-associating species, and also the hydrodynamic concentration dependence parameter,g or $\text{g}$, can be evaluated. Using two different models for the hydrodynamic concentration parameter, four different methods are presented for the evaluation of the s_i's. Methods for evaluating g or $\text{g}$, once the s_i's are known, are presented. A method for obtaining the number average sedimentation coefficient, s_N, and its application to self-associations is presented. Methods are shown for the evaluation of Z average properties, x_zc, as well as number average properties,x_Nc, of a self-associating solute from its weight average properties, x_wc.     

Covalent binding of proteins and glucose-6-phosphate dehydrogenase to cellulosic carriers activated with s-triazine trichloride

A method was developed for covalently binding proteins and enzymes to cellulosic carriers such that the enzymes retained high specific activity. Optimal conditions for activating the carriers with s-triazine trichloride were found to be: (a) pretreatment of cellulose with 3 m NaOH; and (b) reaction with 5% ($\text{w}\text{w}$) s-triazine trichloride in dioxane-xylene (1:1 $\text{w}\text{w}$) for 30 min at room temperature. All proteins tested bound most readily at pH values below pH 7. Extensive investigation of immobilized glucose-6-phosphate dehydrogenase showed that: (a) over 80% of the specific activity of the enzyme was retained; and (b) the pH optimum and K_m values were not altered significantly from that of the free enzyme. The binding method has been applied successfully to hexokinase, phosphorylase and pronase.     

Equilibrium centrifugation of proteins in acidic solutions. Beta-lactoglobulin A in aqueous acetic, propionic, and butyric acids.

Short chain aliphatic acids are almost neutrally buoyant in aqueous solutions, and preferential interaction of macromolecules with these solvent components should not greatly affect apparent molecular weights determined by equilibrium ultracentrifugation. The feasibility of molecular weight estimations using native, neutral pH values of partial specific volume has been tested: equilibrium ultracentrifugation of β-lactoglobulin A (β-LgA) has been carried out in aqueous acetic, propionic, and butyric acids in the absence of any other added electrolyte. These solutions are highly nonideal because of the extreme Donnan effect. Apparent molecular weights estimated at infinite dilution using the native neutral pH value of the partial specific volume, $\text{v}{}_{\mathrm{p}}$, differed by less than 5% from the monomer formula weight. The 10 m acids appear to be least effective as dissociating agents for β-LgA, with a weak reversible monomer-dimer association suggested in 10 m acetic acid, with significant heterogeneity apparent in 10 m propionic acid, and with a lack of direct solubility in 10 m butyric acid. All the 0.1 m acids and all the 1 m acids were essentially equally effective as dissociating agents, with the exception of 1 m butyric acid which dissolved β-LgA only slowly to give significantly heterogeneous solutions. From these results and from our previous experiments with aldolase (6), it appears feasible to use the native values of $\text{v}{}_{\mathrm{p}}$ to obtain estimates of molecular weights of proteins in aqueous organic acids as dissociating agents.     

Proteins in the nervous system — structure and function : Progress in clinical and biological research; volume 79

We have purified a unique enzyme, α-amino-?-caprolactam racemase 945-fold from an extract of Achromobacter obae by Octyl—Sepharose CL-4B and Thiopropyl—Sepharose 6B and some other chromatographies. The purified enzyme was found homogeneous by sodium dodecyl sulfate—polyacrylamide gel electrophoresis and analytical ultracentrifugation. The enzyme has a monomeric structure with M_r ～ 50 000 and a sedimentation coefficient (s_20,w) of 4.28 S. The enzyme contains pyridoxal 5'-phosphate as a coenzyme. The pH optimum for the enzyme activity is ～9.0. D- and L-α-amino-?-caprolactams are the only substrates. The K_m values for the D- and L-isomers are, 8 and 6 mM, respectively.