Alteration of the substrate specificity of mouse 2A P450s by the identity of residue-209: steroid-binding site and orientation

Mouse steroid 7- and 15-hydroxylases (P450c7 and P450c15) and coumarin 7-hydroxylase (P450coh) are structurally similar. To study the structural basis of the substrate specificities of these enzymes, we constructed a series of the mutant P450s, expressed in COS-1 and yeast cells, and studied them spectroscopically as well as enzyme-kinetically. A single amino acid mutation of residue-209 is sufficient to alter the substrate specificity of the P450s from xenobiotics to steroids and subsequently, from testosterone to corticosterone. Moreover, residue-209, when it is asparagine, appears to bind directly to the 11β-hydroxyl of corticosterone. The mutations also after the spin equilibrium of P450 depending on the hydrophobicity and size of residue-209. We conclude, therefore, that residue-209 resides close to the 6th ligand of heme in the mouse 2A subfamily and is located at a critical site of the substrate-binding pocket. As a result, the identity of the residue-209 plays a key role in determining the substrate specificity.     

Alteration of high and low spin equilibrium by a single mutation of amino acid 209 in mouse cytochromes P450

The identities of the amino acid at position 209 are most critical in determining specific coumarin 7- and steroid 15 alpha-hydroxylase activity in P450coh and P450(15)alpha, respectively. This system, therefore, provides us with an excellent model to study the structural basis for P450 specificity as a monooxygenase. We expressed in Saccharomyces cerevisiae a series of the mutated P450s in which residue 209 was substituted with the various amino acids and characterized the spectral property and hydroxylase activity of these mutated P450s. The positioning of a hydrophobic residue including Phe, Leu, and Val at position 209 resulted in shifting the P450 to the high-spin state, while a charged amino acid such as Lys or Asp produced the low-spin form. Moreover, a P450 with Asn or Gly in this position exhibited spectra indicating a mixture of the high- and low-spin forms. This spin alteration, depending upon the hydrophobicity and size of residue at position 209, indicates that this position is likely to reside close to the sixth axial ligand on the distal surface of the heme in these P450s. This proximity of residue 209 to the ligand may explain the critical role of this residue in determining the hydroxylase specificity and activity of these P450s.     

Roles of residues 129 and 209 in the alteration by cytochrome b5 of hydroxylase activities in mouse 2A P450S.

Cytochrome b5 stimulates the coumarin 7-hydroxylation activity of P450coh. A mutation of Arg-129 in P450coh, however, abolishes the stimulation. Moreover, this mutant P450coh binds loosely to cytochrome b5-conjugated Sepharose 4B, whereas wild-type P450coh binds tightly. Consistent with this, the mutation increases the Ka value for b5 binding approximately 6-fold. The identity of residue 209 also alters the stimulation of the activity of P450coh depending on the type of the substrates used and products formed. Coumarin 7-hydroxylation activity is greatly stimulated by cytochrome b5 only when Phe is at position 209, while cytochrome b5 stimulates testosterone hydroxylation activity of P450coh in which Phe, Asn, Ser or Lys substitutes residue 209. P450coh changes its rate of hydrogen peroxide formation depending on the identity of residue 209 and substrate used. Cytochrome b5 decreases the hydrogen peroxide formation of some P450coh whose activities are stimulated by the cytochrome; however, the decrease does not always result in stimulating the activity. The results indicate, therefore, that residues 129 and 209 play different roles in stimulating P450coh activity by cytochrome b5; Arg-129 is a key residue in the cytochrome b5-binding domain and is essential for the stimulation. Residue 209, however, alters the efficiency of electron transport for substrate oxidation as a residue which resides near the sixth ligand of heme and in the substrate-binding site.     

Mouse steroid 15 alpha-hydroxylase gene family: identification of type II P-450(15)alpha as coumarin 7-hydroxylase

We identified type II P-450(15)alpha as mouse coumarin 7-hydroxylase (P-450coh). Unlike type I P-450(15)alpha, the other member within the mouse steroid 15 alpha-hydroxylase gene family, type II catalyzed little steroid 15 alpha-hydroxylase activity, yet structurally there were only 11 substitutions between type I and type II P-450(15)alphaS within their 494 amino acid residues (Lindberg et al., 1989), and the N-terminal sequence (21 residues) of P-450coh was identical with that of both P-450(15)alphaS. Induction by pyrazole of coumarin 7-hydroxylase activity correlated well with the increase of type II P-450(15)alpha mRNA in 129/J male and female mice. Pyrazole, on the other hand, was less in males or not effective in females in inducing the 15 alpha-hydroxylase activity and type I P-450(15)alpha mRNA. Expression of type I and II in COS-1 cells revealed that the latter catalyzed coumarin 7-hydroxylase activity at 10 to approximately 14 pmol min-1 (mg of cellular protein)-1. The former, on the other hand, had a high testosterone 15 alpha-hydroxylase but little coumarin 7-hydroxylase activity. It was concluded, therefore, that type II P-450(15)alpha is the mouse coumarin 7-hydroxylase. Identification of type II as the P-450 specific to coumarin 7-hydroxylase activity and characterization of its cDNA and gene, therefore, were significant advances toward understanding the basis of genetic regulation of this activity in mice (known as Coh locus).     

Mouse liver P450Coh: genetic regulation of the pyrazole-inducible enzyme and comparison with other P450 isoenzymes

Genetic experiments with two inbred strains of mice, AKR/J and DBA/2N, show a single major gene inheritance of additive mode for pyrazole-inducible coumarin 7-hydroxylase. Intragroup variation in the enzyme activity further suggests the contribution of minor modifying genes to the final enzyme activity. Western blot analysis with a polyclonal antibody raised against the purified isozyme P450Coh (highly active in the 7-hydroxylation of coumarin) showed that a difference in the amounts of P450Coh protein between the D2 and AKR mice is the reason for the differences in the enzyme activity between the two mouse strains. Accordingly, changes at the regulatory level rather than at the structural gene would explain the genetic difference in the activity of coumarin 7-hydroxylase. This hypothesis is further supported by the identical Km values of the basal and induced enzyme. The inducibility of coumarin 7-hydroxylase by phenobarbital (PB) and its genetic regulation have been previously studied by A. W. Wood and colleagues ((1974) Science 185, 612-614; (1979); J. Biol. Chem. 254, 5641-5646 and 5647-5651). Our present experiments show that the regulation is the same for the pyrazole-inducible enzyme. Furthermore the experiments with anti-P450Coh antibody show that the PB- and pyrazole-inducible proteins have the same molecular weight and are immunologically indistinguishable. This suggests that PB and pyrazole may induce the same enzyme. Immunoinhibition of microsomal coumarin 7-hydroxylase is practically 100% for control animals and after pretreatment with pyrazole or PB. This suggests that in each case the same or immunologically closely related proteins are metabolizing coumarin and that the P450Coh may be the only P450 isoenzyme in mouse liver microsomes catalyzing the 7-hydroxylation of coumarin. The N-terminal amino acid sequence of P450Coh was found to be identical with those from Type I and Type II genes of the mouse P45015 alpha family for the first 21 amino acids. With rat PB-inducible P450b the homology is only 33%. Also the immunological properties of P450Coh are different from those of P450b. This may suggest that P450Coh has a closer association to the steroid 15 alpha-hydroxylase gene family than to the P450IIB subfamily of phenobarbital-inducible isoenzymes.     

Characterization and regulation of sex-specific mouse steroid hydroxylase genes

We characterized the genes of the male-specific mouse steroid 16 alpha-hydroxylase (C-P-45016 alpha) and the female-specific mouse steroid 15 alpha-hydroxylase (P-45015 alpha) within two distinct gene families. In spite of the high structural identities within each family, the expression of the hydroxylase genes is uniquely regulated. Moreover, the other family members encode the P-450s which are structurally very similar to the hydroxylases but are not able to catalyze steroid hydroxylase activities. For example, only a single amino acid substitution creates steroid 15 alpha-hydroxylase activity in another family-member P-450coh, which catalyzes coumarin 7-hydroxylase but little steroid hydroxylase activity. It appears, therefore, that the mouse P-450 gene families evolved through gene duplication and selective mutation to create new P-450s structurally as well as to establish novel regulatory elements for the gene expressions.     

Analysis of coumarin 7-hydroxylation activity of cytochrome P450 2A6 using random mutagenesis

Cytochrome P450 (P450) 2A6 is an important human enzyme involved in the metabolism of many xenobiotic chemicals including coumarin, indole, nicotine, and carcinogenic nitrosamines. A combination of random mutagenesis and high-throughput screening was used in the analysis of P450 2A6, utilizing a fluorescent coumarin 7-hydroxylation assay. The steady-state kinetic parameters (k(cat) and Km) for coumarin 7-hydroxylation by wild-type P450 2A6 and 35 selected mutants were measured and indicated that mutants throughout the coding region can have effects on activity. Five mutants showing decreased catalytic efficiency (k(cat)/Km) were further analyzed for substrate selectivity and binding affinities and showed reduced catalytic activities for 7-methoxycoumarin O-demethylation, tert-butyl methyl ether O-demethylation, and indole 3-hydroxylation. All mutants except one (K476E) showed decreased coumarin binding affinities (and also higher Km values), indicating that this is a major basis for the decreased enzymatic activities. A recent x-ray crystal structure of P450 2A6 bound to coumarin (Yano, J. K., Hsu, M. H., Griffin, K. J., Stout, C. D., and Johnson, E. F. (2005) Nat. Struct. Mol. Biol. 12, 822-823) indicates that the recovered A481T and N297S mutations appear to be close to coumarin, suggesting direct perturbation of substrate interaction. The decreased enzymatic activity of the K476E mutant was associated with decreases both in NADPH oxidation and the reduction rate of the ferric P450 2A6-coumarin complex. The attenuation is caused in part to lower binding affinity for NADPH-P450 reductase, but the K476E mutant did not achieve the wild-type coumarin 7-hydroxylation activity even at high reductase concentrations.     

Cytochrome P450 isozymes catalyzing 4-hydroxylation of parkinsonism-related compound 1,2,3,4-tetrahydroisoquinoline in rat liver microsomes.

Microsomal 4-hydroxylase of 1,2,3,4-tetrahydroisoquinoline (TIQ), a possible candidate for causing Parkinson disease, was characterized by using rat hepatic microsomes and purified P450 isozymes. Kinetic analysis revealed that Km and Vmax values (mean +/- SE) for hepatic microsomal TIQ 4-hydroxylase of male Wistar rats were 319.6 +/- 26.8 microM and 12.13 +/- 1.43 pmol.min-1.mg-1 protein, respectively. When TIQ 4-hydroxylase activity was compared in Wistar (an animal model of extensive debrisoquine metabolizers) and Dark Agouti (an animal model of poor debrisoquine metabolizers) rats, significant strain (Wistar greater than Dark Agouti) and sex (male greater than female) differences were observed. The microsomal activity toward TIQ 4-hydroxylation was increased by pretreatment of male Wistar rats with P448 inducers (beta-naphthoflavone and sudan I), but not with phenobarbital. Pretreatment with propranolol, an inhibitor of P450 isozymes belonging to the P450 IID gene subfamily, decreased TIQ 4-hydroxylase activity. P450 BTL, a P450 isozyme belonging to the IID subfamily, showed TIQ 4-hydroxylase activity of 64.1 pmol.min-1.nmol P450(-1), which was 3.2-fold that of microsomes (20.9 pmol.min-1.nmol P450(-1)). Antibody (IgG) against this isozyme suppressed microsomal TIQ 4-hydroxylase activity concentration-dependently. A male-specific P450 ml (P450IIC11) catalyzed this reaction to a much lesser extent (10.0 pmol.min-1.nmol P450(-1)), and its antibody did not affect the microsomal activity. These results suggest that TIQ 4-hydroxylation in hepatic microsomes are catalyzed predominantly by a P450 isozyme (or isozymes) belonging to the IID gene subfamily in non-treated rats and its immunochemically related P450 isozyme (or isozymes), and that a P450 isozyme (or isozymes) belonging to the IA subfamily also participates in TIQ 4-hydroxylation in rats pretreated with P448-inducers.     

Interaction of coumarin-hydroxylating cytochrome P-450coh from liver microsomes of mice induced by pyrazole with cytochrome b5

Cytochrome P-450coh from pyrazole-treated mice was shown to form a tight and specific complex with cytochrome b5 from mouse liver microsomes. The complex formation was found to result in type I spectral changes indicating a spin shift from the low to the high spin form. When added to a reconstituted system containing cytochrome P-450coh, NADPH-cytochrome P-450 reductase and phospholipid, cytochrome b5 stimulates hydroxylation of coumarin and O-deethylation of 7-ethoxycoumarin. The maximal stimulating effect is reached at a 1:1 stoichiometry. Mouse liver cytochrome b5 stimulates hydroxylation and deethylation by 100% and 60%, respectively. The stimulating effect of cytochrome b5 was found to result from the increase of the maximal rate of oxidation, being practically without effect on Km. Cytochrome b5 purified from rat and rabbit liver microsomes interacts with cytochrome P-450coh but fails to stimulate the oxidation reaction. At large excess, cytochrome b5 inhibits the oxidations catalyzed by cytochrome P-450coh. Immobilized cytochrome b5 either from mouse or rat and rabbit microsomes proved to be an efficient affinity matrix for cytochrome P-450coh purification.     

Mouse liver phenobarbital-inducible P450 system: purification, characterization, and differential inducibility of four cytochrome P450 isozymes from D2 mouse

Three novel cytochrome P450 isozymes were purified from phenobarbital (PB)-treated D2 mouse liver microsomes and compared to the previously characterized coumarin 7-hydroxylase, P450Coh. The molecular masses were 56.5, 55, 51, and 49.5 kDa, and the peaks of the reduced CO complexes were at 450, 447.5, 451.5, and 449 nm for P450PBI, P450PBII, P450PBIII, and P450Coh, respectively. The NH2-terminal sequences suggest that these isozymes belong to the P450 gene subfamilies 2B, 1A, 2C, and 2A, respectively. On the basis of reconstituted activities and microsomal immunoinhibition studies, P450Coh was the sole catalyst of coumarin 7-hydroxylation. P450PBI was the major isozyme catalyzing the high Km 7-pentoxyresorufin O-dealkylation. This reaction was also mediated at a slower rate by the low Km isozyme, P450PBII. P450PBIII contributed significantly to the microsomal O-deethylation of 7-ethoxyresorufin and N-demethylation of benzphetamine. Western blotting and dot immunobinding analyse of microsomes showed that the induction patterns of the isozymes were different. PB and TCPO-BOP induced all isozymes variably: P450PBI (19- and 31-fold), P450PBII (2- and 3-fold), P450PBIII (9- and 4-fold), and P450Coh (about 2-fold). Pyrazole induced only P450Coh, while all other isozymes were decreased by 30 to 60%. The changes in the microsomal amounts of these isozymes correlated generally well with the variation in the immunoinhibitable enzyme activities. On the basis of the structural and catalytic properties, immunochemical characteristics, and induction profiles, all three isozymes were different from each other and from the previously characterized P450Coh. This mouse PB-inducible P450 model may be valuable in further studies on the induction mechanisms of PB and TCPOBOP.     

Kinetics of liver microsomal cholecalciferol 25-hydroxylase in vitamin D-depleted and -repleated rats.

Kinetics of vitamin D-depleted and -repleted rat liver microsomal cholecalciferol 25-hydroxylase were studied. Anaerobiosis, CO, omission of a NADPH-generating system and addition of detergents all decreased the activities, showing that the hydroxylase behaves like a cytochrome P-450-dependent enzyme. An apparent Km of 0.18 micrometer and Vmax. of 32pmol/min per g of tissue were found for vitamin D-deficient animals. Although both apparent Km and Vmax. were significantly altered in vitamin D-repleted animals no inhibition of the enzyme was elicited. These latter results show that at normal vitamin D intake, rat liver cholecalciferol 25-hydroxylase is not feedback-inhibited.     

Catalytic properties of the human cytochrome P450 2E1 produced by cDNA expression in mammalian cells.

A full-length cDNA encoding human cytochrome P450 2E1 was expressed in mammalian cell lines using the vaccinia virus expression system. Immunoblot analysis showed that the expressed protein reacted with a polyclonal antibody against rat 2E1 and comigrated with P450 2E1 from human liver microsomes. P450 2E1 expressed in Hep G2 cells, a human cell line which contains both cytochrome b5 and NADPH:P450 oxidoreductase, was able to metabolize several known P450 2E1 substrates: N-nitrosodimethylamine (NDMA), N-nitrosomethylbenzylamine (NMBzA), p-nitrophenol, phenol, and acetaminophen. Apparent Km and Vmax values for NDMA demethylation were 22 microM and 173 pmol/min/mg microsomal protein, respectively. P450 2E1 expressed in TK-143 cells, which do not contain b5, displayed Km and Vmax values of 31 microM and 34 pmol/min/mg microsomal protein, respectively. Incorporation of purified rat liver b5 into TK-143 microsomes increased the Vmax 2.2-fold and decreased the Km to 22 microM. Addition of b5 to Hep G2 microsomes resulted in a 1.6-fold increase in Vmax, but showed no effect on the Km. P450 2E1 expressed in Hep G2 cells was shown to metabolize NMBzA with a Km of 47 microM and Vmax of 213 pmol/min/mg microsomal protein. Addition of b5 lowered the Km to 27 microM, but had no effect on Vmax. These results demonstrate conclusively that P450 2E1 is responsible for the low Km forms of NDMA demethylase and NMBzA debenzylase observed in liver microsomes and that these activities are affected by cytochrome b5.     

Identification of human cytochrome P450 isoforms involved in the 7-hydroxylation of chlorpromazine by human liver microsomes

Studies to identify the cytochrome P450 (CYP) isoform(s) involved in chlorpromazine 7-hydroxylation were performed using human liver microsomes and cDNA-expressed human CYPs. The kinetics of chlorpromazine 7-hydroxylation in human liver microsomes showed a simple Michaelis-Menten behavior. The apparent Km and Vmax values were 3.4+/-1.0 microM and 200.5+/-83.7 pmol/min/mg, respectively. The chlorpromazine 7-hydroxylase activity in human liver microsomes showed good correlations with desipramine 2-hydroxylase activity (r = 0.763, p < 0.05), a marker activity for CYP2D6, and phenacetin O-deethylase activity (r = 0.638, p < 0.05), a marker activity for CYP1A2. Quinidine (an inhibitor of CYP2D6) completely inhibited while alpha-naphthoflavone (an inhibitor of CYP1A2) marginally inhibited the chlorpromazine 7-hydroxylase activity in a human liver microsomal sample showing high CYP2D6 activity. On the other hand, alpha-naphthoflavone inhibited the chlorpromazine 7-hydroxylase activity to 55-65% of control in a human liver microsomal sample showing low CYP2D6 activity. Among eleven cDNA-expressed CYPs studied, CYP2D6 and CYP1A2 exhibited significant activity for the chlorpromazine 7-hydroxylation. The Km values for the chlorpromazine 7-hydroxylation of both cDNA-expressed CYP2D6 and CYP1A2 were in agreement with the Km values of human liver microsomes. These results suggest that chlorpromazine 7-hydroxylation is catalyzed mainly by CYP2D6 and partially by CYP1A2.     

The CYP2A3 gene product catalyzes coumarin 7-hydroxylation in human liver microsomes

Three cDNAs, designated IIA3, IIA3v, and IIA4, coding for P450s in the CYP2A gene subfamily were isolated from a lambda gt11 library prepared from human hepatic mRNA. Only three nucleotide differences and a single amino acid difference, Leu160----His, were found between IIA3 and IIA3v, indicating that they are probably allelic variants. IIA4 displayed 94% amino acid similarity with IIA3 and IIA3v. The three cDNAs were inserted into vaccinia virus, and recombinant viruses were used to infect human hepatoma Hep G2 cells. Only IIA3 was able to produce an enzyme that had a reduced CO-bound spectrum with a lambda max at 450 nm. This expressed enzyme was able to carry out coumarin 7-hydroxylation (turnover number of 15 min-1) and ethoxycoumarin O-deethylation. cDNA-expressed IIA3v and IIA4 failed to incorporate heme and were enzymatically inactive. Analysis of IIA proteins in human liver microsomes, using antibody against rat IIA2, revealed two proteins of 49 and 50 kDa, the former of which appeared to correlate with human microsomal coumarin 7-hydroxylase activity. A more striking correlation was found between IIA mRNA and enzyme activity. The rat antibody was able to completely abolish coumarin 7-hydroxylase activity in 12 liver samples. In addition, kinetics of coumarin metabolism in two livers were monophasic over the substrate concentration tested. Km values obtained from human liver (2.3 microM) were similar to those obtained from lysates of hepatoma cells expressing IIA3 (3.6-7.1 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of cytochrome P450 2A4 and 2A5-catalyzed 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) metabolism

The tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), is a potent lung carcinogen in the A/J mouse, and is believed to be a causative agent for human lung cancer. NNK requires metabolic activation by alpha-hydroxylation to exert its carcinogenic potential. The human P450, 2A6 is a catalyst of this reaction. There are two closely related enzymes in the mouse, P450 2A4 and 2A5, which differ from each other by only 11 amino acids. In the present study these two mouse P450s were expressed in Spodoptera frugiperda (Sf9) cells using recombinant baculovirus. The catalysis of NNK metabolism by Sf9 microsomal fractions containing either P450 2A4 or 2A5 was determined. Both enzymes catalyzed the alpha-hydroxylation of NNK but with strikingly different efficiencies and specificities. P450 2A5 preferentially catalyzed NNK methyl hydroxylation, while P450 2A4 preferentially catalyzed methylene hydroxylation. The KM and Vmax for the former were 1.5 microM and 4.0 nmol/min/nmol P450, respectively, and for the latter 3.9 mM and 190 nmol/min/nmol P450. The mouse coumarin 7-hydroxylase, P450 2A5 is a significantly better catalyst of NNK alpha-hydroxylation than is the closely related human enzyme, P450 2A6.     

Purification and characterization of a microsomal cytochrome P-450 with high activity of coumarin 7-hydroxylase from mouse liver

Phenobarbital-induced coumarin 7-hydroxylase is high in DBA/2J and low in C57BL/6N inbred mice; this genetic difference is encoded by the Coh locus on chromosome 7. The aim of this study was to develop an antibody specific for this cytochrome P-450 polymorphism. P-450 fractions, highly specific for phenobarbital-inducible coumarin 7-hydroxylase activity, were purified from DBA/2J and C57BL/6N mouse liver microsomes. Both proteins are 49 kDa, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Soret peaks of the reduced cytochrome . CO complexes are 451 nm. Reconstituted DBA/2J coumarin 7-hydroxylase activity exhibits a V twice as high as, and a Km value 10-fold less than, the reconstituted C57BL/6N activity. Antibodies were raised in rabbit. By Ouchterlony immunodiffusion, both antibodies show 100% cross-reactivity with DBA/2J and C57BL/6N microsomes and purified antigens. Yet, DBA/2J but not C57BL/6N 7-hydroxylase activity is inhibited by the antibody to DBA/2J P-450. Both DBA/2J and C57BL/6N activities are blocked by the antibody to C57BL/6N P-450. Neither antibody has any effect on liver microsomal d-benzphetamine N-demethylase, ethylmorphine N-demethylase, aminopyrine N-demethylase, 7-ethoxycoumarin O-deethylase, acetanilide 4-hydroxylase, or aryl hydrocarbon (benzo[a]pyrene) hydroxylase activity. The DBA/2J protein most specific for phenobarbital-induced coumarin 7-hydroxylation is designated 'P-450Coh'. Anti-(P-450Coh) precipitates a relatively minor 49-kDa protein from detergent-solubilized microsomes and from in vitro translation of poly(A+)-enriched total RNA of phenobarbital-treated DBA/2J mouse liver, whereas the major phenobarbital-induced P-450 proteins exhibit a molecular mass of about 51 kDa. The immunoprecipitated translation products correspond to a messenger RNA of 2100 +/- 100 nucleotides.     

Phosphorylation of synthetic peptides by skeletal muscle myosin light chain kinases

Substrate determinants for rabbit and chicken skeletal muscle myosin light chain kinases were examined with synthetic peptides. Both skeletal muscle myosin light chain kinases had similar phosphorylation kinetics with synthetic peptide substrates. Average kinetic constants for skeletal muscle myosin light chain heptadecapeptide, (formula; see text) where S(P) is phosphoserine, were Km, 2.3 microM and Vmax, 0.9 mumol/min/mg of enzyme. Km values were 122 and 162 microM for skeletal muscle peptides containing A-A for basic residues at positions 2-3 and 6-7, respectively. Average kinetic constants for smooth muscle myosin light chain peptide, (formula; see text), were Km, 1.4 microM and Vmax 27 mumol/min/mg of enzyme. Average Km values for the smooth muscle peptide, residues 11-23, were 10 microM which increased 6- and 11-fold with substitutions of alanine at residues 12 and 13, respectively. Vmax values decreased and Km values increased markedly by substitution of residue 16 with glutamate in the 11-23 smooth muscle tridecapeptide. Basic residues located 3 and 6-7 residues toward the NH2 terminus from phosphoserine in smooth muscle myosin light chain and 6-8 and 10-11 residues toward the NH2 terminus from phosphoserine in skeletal muscle myosin light chain appear to be important substrate determinants for skeletal muscle myosin light chain kinases. These properties are different from myosin light chain kinase from smooth muscle.     

Benzo(a)pyrene activation to 7,8-dihydrodiol 9,10-oxide by rat liver microsomes. Control by selective product inhibition

Metabolism of benzo(a)pyrene (BP) and 7,8-dihydrodiol by 3-methylcholanthrene (MC)-induced rat liver microsomes are both subject to severe inhibition by primary metabolites of BP, which was analyzed by determining individual inhibition constants for all primary BP metabolites for both BP and 7,8-dihydrodiol metabolism. Monooxygenation of 7,8-dihydrodiol was, surprisingly, 5 to 10 times more sensitive than monooxygenation of BP to inhibition by all primary metabolites, even though both reactions require the same enzyme, cytochrome P-450c. Two representative products, 1,6-quinone and 9-phenol, were both strong, competitive inhibitors of BP metabolism with Ki values of 0.12 and 0.74 microM, respectively. The total effect of product inhibition on the overall reactions was determined by fitting progress curves of BP, 7,8-dihydrodiol, and anti-7,8-dihydrodiol 9,10-oxide (determined as 7,10/8,9-tetrol) over a range of BP concentrations to integrated steady-state equations using experimental Vmax and Km values. The effective product inhibition factors for BP and 7,8-dihydrodiol metabolism, determined from progress curve fits, were only 2-fold higher than the corresponding calculated theoretical values. The effective product inhibition factors, obtained from progress curve analysis, confirmed that 7,8-dihydrodiol metabolism was substantially more sensitive to inhibition by primary BP metabolites than BP metabolism itself. This difference probably reflects the much higher affinity of cytochrome P-450c for BP (Kd = 6 nM), as compared to 7,8-dihydrodiol (Kd = 175 nM) that was established spectrophotometrically both for the purified cytochrome and for MC microsomes. The Km for BP metabolism is 50 to 100 times higher than the Kd, while the Km is similar to the Kd for 7,8-dihydrodiol metabolism. The discrepancy for BP between Km and Kd suggests that standard Michaelis-Menten kinetics may be perturbed by either slow substrate or product dissociation.     

Modulation of the catalytic activity of cruzipain, the major cysteine proteinase from Trypanosoma cruzi, by temperature and pH.

Cysteine proteinases are relevant to several aspects of the parasite life cycle and of parasite-host relationships. Here, a quantitative investigation of the effect of temperature and pH on the total substrate inhibition of cruzipain, the major papain-like cysteine proteinase from Trypanosoma cruzi, is reported. Values of the apparent catalytic and inhibition parameters Km, Vmax, Vmax/Km, and K(i) for the cruzipain-catalysed hydrolysis of N-alpha-benzyloxycarbonyl-L-phenylalanyl-L-arginine-(7-amino-4-methylcoumarin) (Z-Phe-Arg-AMC) and azocasein were determined between 10.0 degrees C and 40.0 degrees C and between pH 4.5 and 8.5. Values of Km were independent of temperature and pH, whereas values of Vmax, Vmax/Km, and K(i) were temperature-dependent and pH-dependent. Over the whole pH range explored, values of logVmax, log(Vmax/Km), and logK(i) increased linearly with respect to T(-1). Values of Vmax and Vmax/Km were affected by the acid-base equilibrium of one temperature-independent ionizing group (i.e. pK(unl)' = pK(lig)' = 5.7 +/- 0.1, at 25.0 degrees C). Moreover, values of K(i) were affected by the alkaline pK shift of one ionizing group of active cruzipain (from pK(unl)" = 5.7 +/- 0.1 to pK(lig)" = 6.1 +/- 0.1, at 25.0 degrees C) upon Z-Phe-Arg-AMC binding. Values of logK(unl)', logK(lig)', and logK(lig)" were temperature-independent. Conversely, values of logK(unl)" were linearly dependent on T(-1). As a whole, total substrate inhibition of cruzipain decreased with increasing temperature and pH. These data suggest that both synthetic and protein substrates can bind to the unique active centre of cruzipain either productively or following a binding mode which results in enzyme inhibition. However, allosteric effect(s) cannot be excluded.