Biphasic kinetic behavior of rat cytochrome P-4501A1-dependent monooxygenation in recombinant yeast microsomes.

Rat cytochrome P-4501A1-dependent monooxygenase activities were examined in detail using recombinant yeast microsomes containing rat cytochrome P-4501A1 and yeast NADPH-P-450 reductase. On 7-ethoxycoumarin, which is one of the most popular substrates of P-4501A1, the relationship between the initial velocity (v) and the substrate concentration ([S]) exhibited non-linear Michaelis-Menten kinetics. Hanes-Woolf plots ([S]/v vs. [S]) clearly showed a biphasic kinetic behavior. Aminopyrine N-demethylation also showed a biphasic kinetics. The regression analyses on the basis of the two-substrate binding model proposed by Korzekwa et al. (Biochemistry 37 (1998) 4137-4147) strongly suggest the presence of the two substrate-binding sites in P-4501A1 molecules for those substrates. An Arrhenius plot with high 7-ethoxycoumarin concentration showed a breakpoint at around 28 degrees C probably due to the change of the rate-limiting step of P-4501A1-dependent 7-ethoxycoumarin O-deethylation. However, the addition of 30% glycerol to the reaction mixture prevented observation of the breakpoint. The methanol used as a solvent of 7-ethoxycoumarin was found to be a non-competitive inhibitor. Based on the inhibition kinetics, the real V(max) value in the absence of methanol was calculated. These results strongly suggest that the recombinant yeast microsomal membrane containing a single P-450 isoform and yeast NADPH-P-450 reductase is quite useful for kinetic studies on P-450-dependent monooxygenation including an exact evaluation of inhibitory effects of organic solvents.

Inhibitory effects of 1,4-naphthoquinone derivatives on rat cytochrome P4501A1-dependent monooxygenase activity in recombinant yeast microsomes.

We reported previously that various naphthoquinone derivatives inhibited cytochrome P450-dependent monooxygenase of liver and placenta microsomes [Muto, N. et al. (1987) Biochem. Biophys. Res. Commun. 146, 487-494]. To understand the complex inhibitory behaviors that were observed, it is desirable to study the relationship between structure and inhibitory activity of naphthoquinones in a simplified system containing a single P450 species. In the present study, the inhibitory effects of six derivatives of 1,4-naphthoquinone (hereafter referred to as NQ) on rat cytochrome P4501A1-dependent 7-ethoxycoumarin O-deethylation were examined using yeast microsomes containing overexpressed rat P4501A1. Of these, 2-methyl-5-hydroxy-NQ, 2-methyl-NQ, 2-hydroxy-NQ, and NQ showed competitive inhibition, whereas 5,8-dihydroxy-NQ and 5-hydroxy-NQ showed noncompetitive inhibition. Judging from the inhibitor constant (K(i)), the binding affinity of the four competitive inhibitors for the substrate-binding pocket of P4501A1 is in the order: 2-CH(3)-5-OH-NQ > 2-CH(3)-NQ > NQ >> 2-OH-NQ. On binding with P4501A1, 2-CH(3)-5-OH-NQ, 2-CH(3)-NQ, and NQ induced distinct Type II, Type I, and reverse Type I spectra, respectively. These results indicate that methyl and hydroxyl groups introduced into NQ have unique effects on their binding mode and binding affinity.

Lysyl-tRNA synthetase of Bacillus stearothermophilus molecular cloning and expression of the gene.

The gene of the lysyl-tRNA synthetase of Bacillus stearothermophilus NCA1503 was cloned and sequenced. The gene consists of 1485 bp nucleotides commencing with an ATG start codon and ending with a TAA stop codon, and encodes a polypeptide of 493 amino acids. The recombinant enzymes were expressed in E. coli using an expression plasmid containing the T7 RNA polymerase/promoter.

Sodium chloride enhances markedly the thermal stability of thermolysin as well as its catalytic activity.

Thermolysin, a thermophilic metalloproteinase, is markedly activated in the presence of high concentrations (1-5 M) of neutral salts. The activity increases in an exponential fashion with increasing salt concentration, and is enhanced 13-15 times with 4 M NaCl at pH 7.0 and 25 degreesC (K. Inouye, Effects of salts on thermolysin: activation of hydrolysis and synthesis of N-carbobenzoxy-l-aspartyl-l-phenylalanine methyl ester, and a unique change in the absorption spectrum of thermolysin, J. Biochem. 112 (1992) 335-340). In this study, the effect of NaCl on the thermal stability of thermolysin has been examined at 60-85 degreesC. The activation energy, Ea, for the thermal inactivation is 15 kcal/mol at 0 M NaCl, and increases up to 30-33 kcal/mol by the addition of 0. 5-1.5 M NaCl. Further increase in [NaCl] decreases the Ea value, and at 4 M NaCl it is almost the same as that at 0 M NaCl. Thermolysin at 0.5-1.5 M NaCl is twice as heat-stable as in the absence of NaCl. The NaCl dependence of the stability is different from that of the activity, suggesting that the effects of NaCl on activity and stability are independent. Thermolysin has been demonstrated to be not only a thermophilic enzyme but also a highly halophilic one.

Effect of salts on the solubility of thermolysin: a remarkable increase in the solubility as well as the activity by the addition of salts without aggregation or dispersion of thermolysin.

Thermolysin is remarkably activated in the presence of high concentrations (1-5 M) of neutral salts [Inouye, K. (1992) J. Biochem. 112, 335-340]. The activity is enhanced 13-15 times with 4 M NaCl at pH 7.0 and 25 degrees C. In this study, the effect of neutral salts on the solubility of thermolysin has been examined. Although the solubility was only 1.0-1.2 mg/ml in 40 mM Tris-HCl buffer, pH 7.5, in the temperature range between 0 and 60 degrees C, it was increased greatly by the addition of salts. With NaCl, the solubility showed a bell-shaped behavior with increasing NaCl concentration, and the maximum solubility (10 mg/ml) was at 2.0-2.5 M NaCl. With LiCl and NaI, it increased progressively to 20-50 mg/ml with increasing salt concentration up to 5 M. The solubility observed in the presence of salts decreased with increasing temperature from 0 to 60 degrees C, and also with the order of chaotropic anion effect. The molecular weight of thermolysin was estimated to be 33.0(+/-2.5) x 10(3) in the presence of 0-3 M NaCl, suggesting that thermolysin exists as a monomer in the presence or absence of 3 M NaCl. The possibility that aggregation and/or dispersion of thermolysin might be related to the remarkable activation by salt was ruled out.

Lysyl-tRNA synthetase from Bacillus stearothermophilus. Stopped-flow kinetic analysis of enzyme.lysyladenylate formation.

Amino acid activation reaction of the lysyl-tRNA synthetase [L-lysine:tRNALys ligase (AMP forming); EC 6.1.1.6] from Bacillus stearothermophilus was studied fluorometrically by the stopped-flow method. The addition of L-lysine to the enzyme solution caused quenching of the protein fluorescence and the subsequent addition of ATP restored the quenched fluorescence [Takita et al. (1996) J. Biochem. 119, 680-689; Takita et al. (1997) 121, 244-250]. In the stopped-flow analysis, however, the former fluorescence change (quenching) could not be detected, while the latter change (restoration) was detectable. The L-lysine binding process was suggested to be much faster than the ATP binding process, being completed within the dead-time of the apparatus, ca. 3 ms. The hyperbolic dependence of kapp on the initial ATP concentration suggested that the ATP binding to the enzyme.L-lysine complex followed a two-step mechanism. Two L-lysine analogues that exhibit the qualitatively similar behavior to L-lysine in the fluorometric titration, L-lysine hydroxamate and L-lysine amide, were examined similarly. The two-step process was also suggested for these analogues, and the forward rate constant in the rate-determining step for L-lysine amide (221+/-7 s-1) was significantly larger than those for L-lysine (45.7+/-4.6 s-1) and L-lysine hydroxamate (14. 5+/-1.7 s-1) at pH 8.0, 30 degrees C.

Effects of nitration and amination of tyrosyl residues in thermolysin on its hydrolytic activity and its remarkable activation by salts.

Thermolysin is remarkably activated in the presence of high concentrations (1-5 M) of neutral salts and its activity is enhanced 15 times by 4 M NaCl at pH 7.0 and 25 degrees C [Inouye, K. (1992) J. Biochem. 112, 335-340]. In this study, the effects of nitration and amination of tyrosyl residues in thermolysin on its halophilic properties were examined. Nitration and successive amination inactivate thermolysin progressively as the degree of modification increases. When 16 tyrosyl residues were nitrated, the activity decreased to 10% of that of the native enzyme, whereas it recovered to 30% when they were aminated. The decrease in the activity by the nitration and amination was shown to be brought about only by a decrease in the molecular activity, kcat; the Michaelis constant, Km, was unaltered. When 14 tyrosyl residues of thermolysin were nitrated, the degree of activation by 4 M NaCl at pH 7.0 decreased from 15 to 10, and this decreased further to 5 when the pH of the reaction medium was raised to 8.5. However, when the nitrated tyrosyl residues were reduced to aminotyrosyl residues, the degree of activation was restored to that of the native enzyme. The change in the degree of activation by nitration and amination of thermolysin could be due to the change in the ionization of tyrosyl residues, and it was suggested that removing negative charges from tyrosyl residues of thermolysin enhances its halophilicity.

Effects of pH, temperature, and alcohols on the remarkable activation of thermolysin by salts.

The activity of thermolysin in the hydrolysis of N-[3-(2-furyl)acryloyl] (FA)-dipeptide amides and N-carbobenzoxyl-L-aspartyl-L-phenylalanine methyl ester is remarkably enhanced by high concentrations (1-5 M) of neutral salts. The activation is due to an increase in the molecular activity, k(cat), while the Michaelis constant, K(m), is not affected by the addition of NaCl. In the present study, the effect of NaCl on the thermolysin-catalyzed hydrolysis of FA-glycyl-L-leucine amide (FAGLA) has been examined by changing the pH and temperature, and by adding alcohols to the reaction mixture. The enzyme activity, expressed by k(cat)/K(m), is pH-dependent, being controlled by two functional residues with pK(a) values of 5.4 and 7.8 in the absence of NaCl. The acidic pK(a) is shifted from 5.4 to 6.7 by the addition of 4 M NaCl, while the basic one is not changed. The degree of activation at a given concentration of NaCl is pH dependent in a bell-shaped manner with the optimum pH around 7. Although the activity increases in both the presence and absence of NaCl with increasing temperature from 5 to 35 degrees C, the degree of activation decreases. Alcohols inhibit thermolysin, and the degree of activation decreases with increasing alcohol concentration. The degree of activation tends to increase with increasing dielectric constant of the medium, although it varies considerably depending on the species of alcohol. Electrostatic interactions on the surface and at the active site of thermolysin are suggested to play a significant role in the remarkable activation by salts.

The states of tyrosyl residues in thermolysin as examined by nitration and pH-dependent ionization.

The states of 28 tyrosyl residues of thermolysin have been characterized by means of pH-jump studies and nitration with tetranitromethane. The ionization states of phenolic groups of the tyrosyl residues have also been estimated by spectrophotometric titration of the absorption change at 295 nm. The ionization of 16 tyrosyl residues was completed within 15 s after a pH-jump, and these residues are considered to be located on the surface of thermolysin. On the other hand, the ionization of the other 12 residues required 15 s to 10 min, suggesting the occurrence of a conformational change which leads to exposure of the buried tyrosyl residues to the solvent. Sixteen tyrosyl residues were nitrated and categorized into three classes according to reactivity. The second-order rate constants of the respective classes of tyrosyl residues for nitration were evaluated as 3.32, 0.52, and 0.18 M-1.min-1, and their apparent pKa values were estimated to be 10.2, 11.4, and 11.8. Tyrosyl residues in the first class were considered to be located almost freely on the surface, while those in the second and third classes might be in constrained states.

Lysyl-tRNA synthetase from Bacillus stearothermophilus. Formation and isolation of an enzyme-lysyladenylate complex and its analogue.

The formation of an enzyme.lysyladenylate complex was studied with a highly purified lysyl-tRNA synthetase [L-lysine:tRNALYS ligase (AMP-forming); EC 6.1.1.6] from Bacillus stearothermophilus. The apparent dissociation equilibrium constants of the enzyme with L-lysine and ATP in the process of the complex formation were estimated to be 50.9 and 15.5 microM, respectively, at pH 8.0, 30 degrees C, by fluorometric measurement. The isolated enzyme.lysyladenylate complex was relatively stable with a rate constant of decomposition of 1.7 x 10(-5) s-1 at pH 8.5 and 0 degree C. The rate constant of transfer of L-lysine from the complex to Escherichia coli tRNA was 1.2 x 10(-2) S-1 at pH 8.5 and 0 degree C. The effects of replacing L-lysine by several analogues on the complex formation were examined. L-Lysine hydroxamate, a strong inhibitor of the L-lysine dependent ATP-PPi exchange reaction, produced a stable complex with the enzyme and ATP, enzyme.lysinehydroxamate-AMP probably being formed. The binding stoichiometry of the assumed L-lysinehydroxamate-AMP per mol of the dimer enzyme was 1:1.

Effect of amino acid residues at the cleavable site of substrates on the remarkable activation of thermolysin by salts.

The activity of thermolysin in the hydrolysis of N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide and N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester is remarkably enhanced in the presence of high concentrations (1-5 M) of neutral salts [Inouye (1992) J. Biochem. (Tokyo) 112, 335-340]. In this study, the effect of salts on such activity has been examined using a series of substrates, furylacryloyl dipeptide amides, which have various hydrophobic amino acids at the cleavable bond. Although the enzyme activity varies widely depending on the substrate employed, the degree of activation at a given concentration of NaCl is considerably similar. This indicates that the degree of activation is not dependent on the hydrophobicity of the amino acid side chains at the scissile bond of the substrates. The molecular activity, kcat, and Michaelis constant, Km, were evaluated separately for substrates N[3-(2-furyl)acryloyl]-L-leucyl-L-alanine amide and N-[3-(2-furyl)acryloyl]L-phenyl-alanyl-L-alanine amide, and the activation was found to be brought about only by an increase in k(cat'). The effectiveness of monovalent cations on the increase of k(cat) was determined to follow the order of Na(+)>K(+)>Li(+).

Lysyl-tRNA synthetase from Bacillus stearothermophilus. Purification, and fluorometric and kinetic analysis of the binding of substrates, L-lysine and ATP.

Lysyl-tRNA synthetase [L-lysine:tRNA(Lys)ligase (AMP forming); EC 6.1.1.6] was purified from Bacillus stearothermophilus NCA1503 approximately 1,100-fold to homogeneity in PAGE. The enzyme is a homodimer of M(r) 57,700 x 2. The molar absorption coefficient, epsilon, at 280 nm is 71,600 M-1.cm-1 at pH8.0. Enzyme activity in the tRNA aminoacylation reaction and the ATP-PPi exchange reaction increases up to 50 degrees C at pH 8.0, but is lost completely at 70 degrees C. The pH-optima of the two reactions are 8.3 at 37 degrees C. In the tRNA aminoacylation reaction, the Km values for L-lysine and ATP are 16.4 and 23.2 muM, respectively, and in the ATP-PPi exchange reaction, the Km values for L-lysine and ATP are 23.6 and 65.1 muM, respectively at 37 degrees C, pH 8.0. Interaction of either L-lysine or ATP with the enzyme has been investigated by using as a probe the ligand-induced quenching of protein fluorescence and by equilibrium dialysis. These static analyses, as well as the kinetic analysis of the L-lysine dependent ATP-PPi exchange reaction indicate that the binding mode of L-lysine and ATP to the enzyme is sequential ordered (L-lysine first). The interaction of lysine analogues with the enzyme has also been investigated.

Effects of alcohols on the hydrolysis of colominic acid catalyzed by Streptococcus neuraminidase.

Regulation of the activity of neuraminidase of Streptococcus sp. (group K) was evaluated by examining the effects of alcohols on the hydrolysis of colominic acids catalyzed by the neuraminidase. Two kinds of alcohol binding site, activation and inhibition sites, were proposed to exist. Competitive inhibition was observed with alcohols smaller than polyethylene glycol #300 (average molecular weight: 300), so the inhibition site is considered to be the substrate binding site, the size of which was estimated to be 10 A in diameter. On the contrary, polyethylene glycols larger than this size activated the enzyme by 1.5-1.8 times. The activity could be raised by binding of the polyethylene glycols to the activation site. This activation was shown to be due solely to the decrease in the Michaelis constant, Km. The smaller polyethylene glycols (#200 and #300) were also considered to bind to the activation site, although activation was not clearly observed due to compensation with inhibition. Strong substrate inhibition by colominic acid was also observed. The activity of Streptococcus neuraminidase was shown to be regulated intricately by the substrate colominic acid and alcohols contained in the reaction medium.

Effect of a p-nitro group of phenyl-maltooligosaccharide substrate on the change of action specificity of lysine-modified porcine pancreatic alpha-amylase.

The effect of chemical modification of lysine residues on the activity of porcine pancreatic alpha-amylase (PPA) was examined, using p-nitrophenyl-alpha-D-maltoside, p-nitrophenyl-alpha-D-maltotrioside, phenyl-alpha-D-maltoside and phenyl-alpha-D-maltotrioside as substrates. Chemical modification of PPA with trinitrobenzenesulfonic acid enhanced the kcat/Km values for p-nitrophenyl substrates, but not for phenyl substrates. Thus, this effect is substituent selective. Considering the productive binding modes of substrates to PPA, the p-nitro group of the substrate and the modified lysine residues of the enzyme would non-ionically interact with each other to stabilize the productive binding mode.

A spectrophotometric study on the interaction of thermolysin with chloride and bromide ions, and the state of tryptophyl residue 115.

The activity of thermolysin is greatly enhanced in the presence of high concentrations of neutral salts [Holmquist, B. and Vallee, B.L. (1976) Biochemistry 15, 101-107; Inouye, K. (1992) J. Biochem. 112, 335-340]. NaBr and NaCl are the most effective for the activation. An absorption difference spectrum with a peak around 293 nm, which is characteristic of the red-shift of a tryptophyl residue caused by charge effects, was observed on mixing of thermolysin with NaCl. As the peak disappeared in the presence of competitive inhibitors of the enzyme (phosphoramidon and zincov), it was considered to be derived from a tryptophyl residue (Trp 115) located in the active site of the enzyme. On the other hand, this peak was not observed on the mixing of thermolysin and NaBr, indicating that the slight difference in size between chloride and bromide ions is critical for the interaction with the tryptophyl residue. NaCl and NaBr exhibit comparable effects on the activation of thermolysin regardless of the considerable discrepancy in their effects on the absorptivity difference around 293 nm. This suggests that the interaction of salts with Trp 115 is not necessarily correlated with the activation of thermolysin.

Analysis of internal motion of single tryptophan in Streptomyces subtilisin inhibitor from its picosecond time-resolved fluorescence.

A mode of internal motion of single tryptophan, Trp 86, of Streptomyces subtilisin inhibitor, was analyzed from its time-resolved fluorescence. The intensity and anisotropy decays of Trp 86 were measured in the picosecond range. These decays were analyzed with theoretical expressions derived assuming that the indole ring of tryptophan as an asymmetric rotor rotates around covalent bonds connecting indole with the peptide chain and an effective quencher of fluorescence of Trp 86 is the nearby SS bond of Cys 35-Cys 50. First, the intensity decays at 6 degrees, 20 degrees, and 40 degrees C were analyzed, and then the both decays of the intensity and anisotropy at 20 degrees C were simultaneously simulated with common parameters. Constants concerning geometrical structures of the protein used for the analysis were obtained from x-ray crystallographic data. Best fit between the observed and calculated decay curves was obtained by a nonlinear least squares method by adjusting a quenching constant averaged over the rotational angles, koq height of the potential energy, p, and three of six diffusion coefficients, Dxx, Dyy, Dzz, Dxy, Dyz, and Dzx, as variable parameters. The obtained results revealed that the internal motion of the indole ring became faster, the quenching rate of the fluorescence of Trp 86 was enhanced and the height of potential energy became lower at higher temperatures, and suggested that Trp 86 was wobbling around the long axis of the indole ring in the protein.

Dissociation of dimer of bovine erythrocyte Cu,Zn-superoxide dismutase and activity of the monomer subunit: effects of urea, temperature, and enzyme concentration.

Cu,Zn-superoxide dismutase (SOD) of bovine erythrocytes is a dimeric enzyme of identical subunits associated through unusually strong noncovalent interactions. It is known not to be dissociated into subunits even in 8 M urea for 72 h at 25 degrees C [Malinowski, D.P. & Fridovich, I. (1979) Biochemistry 18, 5055-5060]. Effects of urea, temperature, and SOD concentration on the inactivation and dissociation into subunits were examined. The activation energy of the inactivation of SOD (at 0.05 mg/ml) was 64 kcal/mol at pH 7.8, and was decreased to 40 kcal/mol in the presence of urea (2.0-7.3 M). The apparent first-order rate constant (kapp) of the inactivation by urea was dependent on the SOD concentration [SOD] during the urea treatment, and SOD showed a higher resistance to the inactivation with increase in the concentration. Dissociation of SOD was monitored by gel filtration HPLC. When SOD solutions of various concentrations were incubated in 6 M urea at 45 degrees C, two monomer species (M1 and M2) were observed in addition to dimer (D). The dimer maintained full activity, while the monomers did not show the activity. The peak areas of these species were changed depending on the SOD concentration during urea treatment; at over 15 mg/ml, almost all SOD was eluted as D, and with a decrease in the SOD concentration, the peak area of D decreased and concomitantly the monomers appeared. M2 could be the sole form in infinitely diluted SOD solution, and D was considered to be converted to M2 through M1. The SOD concentration giving 50% D ([SOD]1/2) was 1.0 mg/ml.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of amino acid residues responsible for the changes of absorption and fluorescence spectra on the binding of subtilisin BPN' and Streptomyces subtilisin inhibitor.

An ultraviolet absorption difference spectrum characteristic of the ionization change of a tyrosyl residue was observed on the binding of subtilisin BPN' with Streptomyces subtilisin inhibitor (SSI) at alkaline pH. This difference spectrum was considered to be induced by a pKa shift (from 9.7 to > or = 11.5) of a tyrosyl residue of subtilisin BPN' in the interaction with carboxyls of SSI [Inouye et al. (1979) J. Biochem. 85, 1115-1126]. In the present paper, the tyrosyl residue in subtilisin BPN' and the carboxyls in SSI were identified by analyzing the difference spectrum using mutants of subtilisin BPN' and SSI: naturally occurring mutants and those prepared by site-directed and cassette mutagenesis. The difference spectrum disappeared on the binding of a mutant subtilisin BPN' of which Tyr104 was replaced by Phe (S-BPN'Y104F) and SSI at pH 9.8. The magnitude of the absorption difference was much smaller when subtilisin BPN' was bound with a mutant SSI of which both Glu67 and Asp68 were replaced by Gly than with the wild-type SSI. These lines of evidence indicated that the difference spectrum was caused by Tyr104 of subtilisin BPN' interacting with Glu67 and Asp68 of SSI. The binding of subtilisin BPN' and SSI is accompanied by an increase of tryptophan fluorescence, which is pH-dependent in the range of pH 7-11 [Uehara et al. (1978) J. Biochem. 84, 1195-1202]. In the present study, this pH-dependence of the fluorescence diminished when SSI bound with S-BPN'Y104F. This suggested that the fluorescence increase was due to Trp106 of subtilisin BPN' and was influenced by the ionization of Tyr104.

Interaction of subtilisin BPN' and recombinant Streptomyces subtilisin inhibitors with substituted P1 site residues.

Kinetic analysis was performed on the interaction between subtilisin BPN' and recombinant species of a proteinaceous proteinase inhibitor, Streptomyces subtilisin inhibitor (SSI), of which the P1 site amino acid residue, Met73, was replaced by site-directed mutagenesis. The inhibitor constant, Ki, was determined from the residual enzyme activity by using a peptide substrate. The rate constant of binding, kon, and the rate constant of dissociation, koff, were determined from a progress curve of the substrate hydrolysis in the presence of the inhibitor by using newly derived equations. A recombinant SSI in which Met73 was replaced by Ile showed an affinity (1/Ki) toward subtilisin BPN' of only about 7% of that of the wild-type SSI, and the kinetic analysis revealed that the increase of koff was responsible for this difference. The affinity of other SSI mutants in which Met73 was replaced by Glu or Asp decreased significantly as pH became increasingly alkaline. The decrease in the affinity of these recombinants was due to the decrease of kon rather than the increase of koff. Stopped-flow studies revealed that the binding reaction was reconcilable with a two-step mechanism, and the kinetic parameters for each step were obtained for the binding of the enzyme and recombinant SSIs.

Change of substrate specificity by chemical modification of lysine residues of porcine pancreatic alpha-amylase.

Lysine residues of porcine pancreatic alpha-amylase (PPA) were modified with trinitrobenzenesulfonate (TNBS). 6 out of 21 lysine residues were modified per PPA molecule. Amylase activity (hydrolysis of the alpha-1,4-D-glucoside bond) was decreased to about 50% of the native enzyme, as judged from the kcat value at pH 6.9 after the modification, whereas maltosidase activity (hydrolysis of p-nitrophenyl-alpha-D-maltoside producing p-nitrophenol and maltose) was increased to about 250%. The increase in maltosidase activity of the modified PPA was due to the increase in kcat, but not to the decrease in Km. Modification of PPA with five kinds of acid anhydrides also caused the same effect as TNBS, including the number of modified lysine residues. The degree of increase in maltosidase activity was fairly proportional to the volume of the incorporated modification reagent. A modification protection study in the presence of maltotriitol (G3OH), which protected two out of six modifiable lysine residues against modification, suggested that a lysine residue at the substrate-binding site contributes to the change of substrate specificity.