Cloning and expression of recombinant human pineal tryptophan hydroxylase in Escherichia coli: purification and characterization of the cloned enzyme.

The first step in the biosynthesis of melatonin in the pineal gland is the hydroxylation of tryptophan to 5-hydroxytryptophan. A cDNA of human tryptophan hydroxylase (TPH) was cloned from a library of human pineal gland and expressed in Escherichia coli. This cDNA sequence is identical to the cDNA sequence published from the human carcinoid tissue [1]. This human pineal hydroxylase gene encodes a protein of 444 amino acids and a molecular mass of 51 kDa estimated for the purified enzyme. Tryptophan hydroxylase from human brainstem exhibits high sequence homology (93% identity) with the human pineal hydroxylase. The recombinant tryptophan hydroxylase exists in solution as tetramers. The expressed human pineal tryptophan hydroxylase has a specific activity of 600 nmol/min/mg when measured in the presence of tetrahydrobiopterin and L-tryptophan. The enzyme catalyzes the hydroxylation of tryptophan and phenylalanine at comparable rates. Phosphorylation of the hydroxylase by protein kinase A or calmodulin-dependent kinase II results in the incorporation of 1 mol of phosphate/mol of subunit, but this degree of phosphorylation leads to only a modest (30%) increase in BH(4)-dependent activity when assayed in the presence of 14-3-3. Rapid scanning ultraviolet spectroscopy has revealed the formation of the transient intermediate compound, 4alpha-hydroxytetrahydrobiopterin, during the hydroxylation of either tryptophan or phenylalanine catalyzed by the recombinant pineal TPH.

Characterization of the electrostatic perturbation of a catalytic site (Cys)-S-/(His)-Im+H ion-pair in one type of serine proteinase architecture by kinetic and computational studies on chemically mutated subtilisin variants.

We have used two structurally well-characterized serine proteinase variants, subtilisins Carlsberg and BPN', to produce (Cys)-S-/(His)-Im+H ion-pairs by chemical mutation in well defined, different, electrostatic microenvironments. These ion-pairs have been characterized by pH-dependent rapid reaction kinetics using, as reactivity probes, thiol-specific time dependent inhibitors, 2,2'-dipyridyl disulfide and 4,4'-dipyrimidyl disulfide, that differ in the protonation states of their leaving groups in acidic media, computer modelling and electrostatic potential calculations. Both ion-pairs possess nucleophilic character, identified by the striking rate maxima in their reactions with 2,2'-dipyridyl disulfide in acid media. In the Carlsberg enzyme, the (Cys220)-S-/(His63)-Im+H ion-pair is produced by protonic dissociation with pKa 4.1 and its reactivity is not perturbed by any detectable electrostatic influence other than the deprotonation of His63 (pKa 10.2). In the BPN' enzyme, the analogous, (Cys221)-S-/(His64)-Im+H ion-pair is produced by protonic dissociation with pKa 5.1 and its reactivity is affected by an ionization with pKa 3.5 in addition to the deprotonation of His64 (pKa > or = 10.35). It is a striking result that calculations using finite difference solutions of the Poisson-Boltzmann equation provide a value of the pKa difference between the two enzyme catalytic sites (0.97) in close agreement with the value (1.0) determined by reactivity probe kinetics when a protein dielectric constant of 2 is assumed and water molecules within 5 A of the catalytic site His residue are included. The pKa difference is calculated to be 0.84 when the water molecules are not included and a protein dielectric constant of 20 is assumed. The calculations also identify Glu156 in the BPN' enzyme (which is Ser in the Carlsberg enzyme) as the main individual source of the pKa shift. The additional kinetically influential pKa of 3.5 is assigned to Glu156 by examining the non-covalent interactions between the 2-pyridyl disulfide reactivity probe and the enzyme active centre region.

Recombinant human phenylalanine hydroxylase: novel regulatory and structural properties.

Recombinant human liver phenylalanine hydroxylase (PAH) expressed in Escherichia coli has been purified to homogeneity. The recombinant enzyme exists in solution as a mixture of 80% tetramers and 20% dimers. A study of the kinetic properties of the enzyme indicates that compared to the recombinant and the native rat liver enzymes, the recombinant human enzyme is in an activated state. This conclusion is supported by the finding that its catalytic activity is only marginally stimulated by incubation with either phenylalanine or lysolecithin. In contrast, the native and the recombinant rat liver enzymes are activated 8- to 25-fold, respectively, when preincubated with phenylalanine or lysolecithin. In the absence of activators, the ratio of the hydroxylase activity in the presence of 6-methyl-5,6,7,8-tetrahydropterin compared to the activity in the presence of (6R)-5,6,7,8-tetrahydrobiopterin (BH4), which is an index of the state of activation of the enzyme, is 4 for the human recombinant PAH compared to a value of 12 for the recombinant rat liver enzyme. Furthermore, the Km for phenylalanine in the presence of BH4 is 0.050 mM, a value that is one-fifth that of the recombinant rat liver enzyme. Covalent modification of the human enzyme by phosphorylation with protein kinase A provides further evidence that the human enzyme is in a substantially activated state. Phosphorylation, which results in the incorporation of 0.6 mol of phosphate/mol of subunit, leads to only a modest activation of 1.5-fold compared to about a 3-fold activation seen after phosphorylation of the native and the recombinant rat liver enzymes. Moreover, the recombinant human liver enzyme is less sensitive than the rat liver enzyme to stimulation by lysolecithin when tryptophan is the substrate. Just as is true for the rat liver enzyme, the apparent Km values for tryptophan and pheylalanine vary with the pterin cofactor employed. The ability of 7-tetrahydrobiopterin (7-BH4) to substitute for the natural cofactor tetrahydrobiopterin has been studied in vitro. The apparent Km for 7-BH4 for the recombinant human enzyme is 0.2 mM and the Km for phenylalanine is 0.05 mM. The hydroxylase reaction is severely inhibited by 7-BH4 in the presence of physiological concentrations of BH4. This inhibition can be overcome by a decrease in the concentration of phenylalanine. The implications of these novel properties of human PAH for phenylalanine homoestasis in man are discussed.

Further studies of the role of Ser-16 in the regulation of the activity of phenylalanine hydroxylase.

It was previously proposed that the activation of rat liver phenylalanine hydroxylase (EC 1.14.16.1) by cAMP-dependent protein kinase-mediated phosphorylation of Ser-16 is due to the introduction of the negatively charged phosphate group. To explore the validity of this proposal, we have applied site-directed mutagenesis to specifically replace Ser-16 with negatively charged amino acids, glutamic and aspartic; with polar uncharged amino acids, asparagine and glutamine; with the positively charged amino acid lysine; and with the nonpolar hydrophobic amino acid alanine. The wild-type and mutant enzymes were purified to homogeneity, and the importance of Ser-16 in the activation of phenylalanine hydroxylase was examined by comparing the state of activation of the phosphorylated form of the wild-type hydroxylase with that of the mutants. The kinetic studies carried out on the wild-type phosphorylated hydroxylase showed that all the activation could be accounted for by an increase in Vmax with no change in Km for either phenylalanine or the pterin cofactor. Replacement of Ser-16 with a negatively charged residue, glutamate of aspartate, resulted in the activation of the hydroxylase by 2- to 4-fold, whereas replacement with glutamine, asparagine, lysine, or alanine resulted in a much more modest increase. Further, lysolecithin was found to stimulate the phosphorylated hydroxylase and the mutant enzymes S16E and S16D by a factor of 6-7. In contrast, the mutants S16Q, S16N, and S16A all showed the same magnitude of activation as the wild-type with lysolecithin. Therefore, this study demonstrates that activation of the enzyme by phosphorylation of Ser-16 by cAMP-dependent protein kinase is due to the introduction of negative charge(s) and strongly suggests the involvement of electrostatic interaction between the regulatory and catalytic domains of the hydroxylase.

Characterization of the wild-type form of 4a-carbinolamine dehydratase and two naturally occurring mutants associated with hyperphenylalaninemia.

The characterization of 4a-carbinolamine dehydratase with the enzymatically synthesized natural substrate revealed non-Michaelis-Menten kinetics. A Hill coefficient of 1.8 indicates that the dehydratase exists as a multisubunit enzyme that shows cooperativity. A mild form of hyperphenylalaninemia with high 7-biopterin levels has been linked to mutations in the human 4a-carbinolamine dehydratase gene. We have now cloned and expressed two mutant forms of the protein based on a patient's DNA sequences. The kinetic parameters of the mutant C82R reveal a 60% decrease in Vmax but no change in Km (approximately 5 microM), suggesting that the cysteine residue is not involved in substrate binding. Its replacement by arginine possibly causes a conformational change in the active center. Like the wild-type enzyme, this mutant is heat stable and forms a tetramer. The susceptibility to proteolysis of C82R, however, is markedly increased in vitro compared with the wild-type protein. We have also observed a decrease in the expression levels of C82R protein in transfected mammalian cells, which could be due to proteolytic instability. The 18-amino acid-truncated mutant GLu-87--> termination could not be completely purified and characterized due to minute levels of expression and its extremely low solubility as a fusion protein. No dehydratase activity was detected in crude extracts from transformed bacteria or transfected mammalian cells. Considering the decrease in specific activity and stability of the mutants, we conclude that the patient probably has less than 10% residual dehydratase activity, which could be responsible for the mild hyperphenylalaninemia and the high 7-biopterin levels.

Purification and cloning of the GTP cyclohydrolase I feedback regulatory protein, GFRP.

The activity of GTP cyclohydrolase I, the initial enzyme of the de novo pathway for biosynthesis of tetrahydrobiopterin, the cofactor required for aromatic amino acid hydroxylations and nitric oxide synthesis, is sensitive to end-product feedback inhibition by tetrahydrobiopterin. This inhibition by tetrahydrobiopterin is mediated by the GTP cyclohydrolase I feedback regulatory protein GFRP, previously named p35 (Harada, T., Kagamiyama, H., and Hatakeyama, K. (1993) Science 260, 1507-1510), and -phenylalanine specifically reverses the tetrahydrobiopterin-dependent inhibition. As a first step in the investigation of the physiological role of this unique mechanism of regulation, a convenient procedure has been developed to co-purify to homogeneity both GTP cyclohydrolase I and GFRP from rat liver. GTP cyclohydrolase I and GFRP exist in a complex which can be bound to a GTP-affinity column from which GTP cyclohydrolase I and GFRP are separately and selectively eluted. GFRP is dissociated from the GTP agarose-bound complex with 0.2 NaCl, a concentration of salt which also effectively blocks the tetrahydrobiopterin-dependent inhibitory activity of GFRP. GTP cyclohydrolase I is then eluted from the GTP-agarose column with GTP. Both GFRP and GTP cyclohydrolase I were then purified separately to near homogeneity by sequential high performance anion exchange and gel filtration chromatography. GFRP was found to have a native molecular mass of 20 kDa and consist of a homodimer of 9.5-kDa subunits. Based on peptide sequences obtained from purified GFRP, oligonucleotides were synthesized and used to clone a cDNA from a rat liver cDNA library by polymerase chain reaction-based methods. The cDNA contained an open reading frame that encoded a novel protein of 84 amino acids (calculated molecular mass 9665 daltons). This protein when expressed in Escherichia coli as a thioredoxin fusion protein had tetrahydrobiopterin-dependent GTP cyclohydrolase I inhibitory activity. Northern blot analysis indicated the presence of an 0.8-kilobase GFRP mRNA in most rat tissues, the amounts generally correlating with levels of GTP cyclohydrolase I and tetrahydrobiopterin. Thus, mRNA levels were relatively high in liver and kidney and somewhat lower in testis, heart, brain, and lung. These results suggest that GFRP is widely expressed and may play a role in regulating not only phenylalanine metabolism in the liver, but also the production of biogenic amine neurotransmitters as well as nitric oxide synthesis.

A re-appraisal of the structural basis of stereochemical recognition in papain. Insensitivity of binding-site-catalytic-site signalling to P2-chirality in a time-dependent inhibition.

1. 2-(N'-Acetyl-D-phenylalanylamino)ethyl 2'-pyridyl disulphide (compound I) [m.p. 123-124 degrees C; [alpha]20D -7.1 degrees (c 0.042 in methanol)] was synthesized, and the results of a study of the pH-dependence of the second-order rate constant (k) for its reaction with the catalytic-site thiol group of papain (EC 3.4.22.2), together with existing kinetic data for the analogous reaction of the L-enantiomer (compound II), were used to evaluate the consequences for transition-state geometry of the difference in chirality at the P2 position of the probe molecule. 2. The kinetic data suggest that the D-enantiomer binds approx. 40-fold less tightly to papain than the L-enantiomer but that the binding-site--catalytic-site signalling that results in a (His-159)-Im(+)-H-assisted transition state occurs equally effectively in the interaction of the former probe as in that of the latter. This results in pH-k profiles for the reactions of both enantiomers each characterized by four macroscopic pKa values (3.7-3.9, 4.1-4.3, 7.9-8.3 and 9.4-9.5) in which k is maximal at pH approx. 6 where the -Im(+)-H-assisted transition state is most fully developed. 3. Model building indicates that both enantiomers can bind to papain such that the phenyl ring of the N-acetylphenylalanyl group makes hydrophobic contacts in the binding pocket of the S2 subsite with preservation of the three hydrogen-bonding interactions involving the substrate analogue reagent and (Asp-158) C = O, (Gly-66) C = O, and (Gly-66)-N-H of papain. Earlier predictions that binding of N-acyl-D-phenylalanine derivatives to papain would be prevented on steric grounds [Berger & Schechter (1970) Philos. Trans. R. Soc. London B 257, 249-264; Lowe & Yuthavong (1971) Biochem. J. 124, 107-115; Lowe (1976) Tetrahedron 32, 291-302] were based on assumed models that are not consistent with the X-ray-diffraction data for papain inhibited by alkylation of Cys-25 with N-benzyloxycarbonyl-Phe-Ala-chloromethane [Drenth, Kalk & Swen (1976) Biochemistry 15, 3731-3738]. 4. The possibility that the kinetic expression of P2-S2 stereospecificity may depend on the nature of the chemistry occurring in the catalytic site of papain is discussed.(ABSTRACT TRUNCATED AT 400 WORDS)

Dependence of the P2-S2 stereochemical selectivity of papain on the nature of the catalytic-site chemistry. Quantification of selectivity in the catalysed hydrolysis of the enantiomeric N-acetylphenylalanylglycine 4-nitroanilides.

1. N-Acetyl-L-phenylalanylglycine 4-nitroanilide and its D-enantiomer were synthesized and characterized and used as substrates with which to evaluate stereochemical selectivity in papain (EC 3.4.22.2)-catalysed hydrolysis. 2. Kinetic analysis at pH 6.0, I 0.1, 8.3% (v/v) NN-dimethylformamide and 25 degrees C by using initial-rate data with [S] much less than Km and weighted non-linear regression provided values of kcat./Km for the catalysed hydrolysis of both enantiomers as (kcat./Km)L = 2040 +/- 48 M-1.S-1 and (kcat./Km)D = 5.9 +/- 0.07 M-1.S-1. These data, taken together with individual values of kcat. and Km for the hydrolysis of the L-enantiomer (a) estimated in the present work as kcat. = 3.2 +/- 1.2 S-1 and Km = 1.5 +/- 0.6 mM and (b) reported by Lowe & Yuthavong [(1971) Biochem. J. 124, 107-115] for the reaction at pH 6.0 in 10% (v/v) NN-dimethylformamide and 35 degrees C, as kcat. = 1.3 +/- 0.2 S-1 and Km = 0.88 +/- 0.1 mM, suggest that (kcat./Km)L congruent to 2000 M-1.S-1 and thus that (kcat./Km)L/(kcat./Km)D congruent to 330.3. Model building indicates that both enantiomeric 4-nitroanilides can bind to papain such that the phenyl ring of the N-acetylphenylalanyl group makes hydrophobic contacts in the S2 subsite with preservation of mechanistically relevant hydrogen-bonding interactions and that the main difference is in the positioning of the beta-methylene group. 4. The dependence of P2-S2 stereochemical selectivity of papain on the nature of the catalytic-site chemistry for reactions involving derivatives of N-acetylphenylalanine is discussed. The variation in the index of stereochemical selectivity (ratio of the appropriate kinetic or thermodynamic parameter for a given pair of enantiomeric ligands), from 330 for the overall acylation process of the catalytic act, through 40 and 31 for the reaction at electrophilic sulphur in 2-pyridyl disulphides respectively without and with assistance by (His-159)-Im(+)-H, to 5 for the formation of thiohemiacetal adducts by reaction at aldehydic carbon, is interpreted in terms of the extent to which conformational variation of the bound ligand in the catalytic-site region permits the binding mode of the -CH2-Ph group of the D-enantiomer to approach that of the L-enantiomer.

Catalytic-site characteristics of the porcine calpain II 80 kDa/18 kDa heterodimer revealed by selective reaction of its essential thiol group with two-hydronic-state time-dependent inhibitors: evidence for a catalytic site Cys/His interactive system and an ionizing modulatory group.

1. Four calpain II heterodimers (80 kDa/30 kDa, 80 kDa/29 kDa, 80 kDa/26 kDa and 80 kDa/18 kDa) were isolated from fresh porcine kidney by (NH4)2SO4 precipitation, chromatography on DEAE-Sepharose CL-6B and subsequently on Reactive Red 120/agarose followed by f.p.l.c. on a Q-Sepharose Hi-Load 16/10 column. 2. The major component (80 kDa/30 kDa) was used to provide the catalytically active calpain II 80 kDa/18 kDa heterodimer by treatment with CaCl2; titration with trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E64) in the presence of monothioglycerol showed the preparation to have 1.0 +/- 0.05 catalytic sites per molecule of heterodimer. 3. The 80 kDa/30 kDa heterodimer was separated from monothioglycerol and other low-molecular-mass material by gel filtration on Sephadex G-25 without loss of catalytic activity towards sulphanilic acid/azocasein in the presence of added Ca2+. On storage overnight at a concentration of 3 microM in KCl at 4 degrees C in the absence of Ca2+ the activator-free preparation still produced fully active 80 kDa/18 kDa heterodimer on addition of Ca2+. 4. Activator-free 80 kDa/30 kDa heterodimer (in the absence of Ca2+) reacts relatively slowly with ethyl 2-pyridyl disulphide at pH 5.9; over 5000 s five thiol groups per molecule react, all at similar rates. In the presence of 8 mM CaCl2 under otherwise identical conditions (and also in the pH range 3.8-10.4) an initial faster phase of reaction corresponding to approx. one thiol group per molecule of heterodimer is generated, but it is not cleanly separated from the subsequent slower reactions on the stopped-flow trace. This fast phase of reaction does not occur when E64-inactivated calpain II is substituted for active 80 kDa/18 kDa heterodimer. 5. Greatly improved resolution of the fast phase of reaction involving the catalytic-site thiol group was achieved by using 2,2'-dipyridyl disulphide (2-Py-S-S-2-Py) instead of ethyl 2-pyridyl disulphide. 6. The pH-dependence of the second-order rate constant (k) for the reaction of the catalytically active activator-free 80 kDa/18 kDa calpain II heterodimer with 2-Py-S-S-2-Py was studied by stopped-flow spectral analysis in the pH range approx. 3-8 without interference from reactions of other thiol groups. 7. The form of the pH-k profile establishes for the first time the existence of an interactive catalytic site system [probably containing a (Cys)-S-/(His)-Im+H ion pair] analogous to those present in monomeric non-Ca(2+)-activated cysteine proteinases.(ABSTRACT TRUNCATED AT 400 WORDS)

Differences in the chemical and catalytic characteristics of two crystallographically 'identical' enzyme catalytic sites. Characterization of actinidin and papain by a combination of pH-dependent substrate catalysis kinetics and reactivity probe studies targeted on the catalytic-site thiol group and its immediate microenvironment.

The characteristics of actinidin (EC 3.4.22.14) and papain (EC 3.4.22.2), two cysteine proteinases whose catalytic-site regions appear to superimpose to a degree that approaches atomic co-ordinate accuracy of both crystal structures, were evaluated by determining (a) the pH-dependence in acid media of the acylation process of the catalytic act (k+2/Ks) using N alpha-benzoyl-L-arginine p-nitroanilide (L-Bz-Arg-Nan) as substrate and (b) the sensitivity of the reactivity of the catalytic-site thiol group and its pH-dependence to structural change in small, thiol-specific, two-protonic-state reactivity probes (2,2'-dipyridyl disulphide and methyl 2-pyridyl disulphide) where enzyme-probe contacts should be restricted to areas close to the catalytic site. Distortion of the catalytic sites of the two enzymes at pH less than 4 was evaluated over time-scales appropriate for both stopped-flow reactivity probe kinetics (less than or equal to 1-2 s) and steady-state substrate catalysis kinetics (3-5 min) by using the 2,2'-dipyridyl disulphide monocation as a titrant for non-distorted catalytic sites. This permitted a lower pH limit to be defined for valid kinetic analysis of both types. The behaviour of the enzymes at pH less than 4 requires a kinetic model in which the apparently biomolecular reaction of enzyme with probe reagent is separated from the process leading to loss of conformational integrity by a potentially reversible step. The acylation of actinidin with L-Bz-Arg-Nan in acidic media occurs in two protonic states, one produced by raising the pH across pKa less than 4 which probably characterizes the formation of -S-/-ImH+ ion pair (pKa approx. 3) and the other, of higher reactivity, produced by raising the pH across pKa 5.5, which may characterize rearrangement of catalytic-site geometry. The pH-dependence of the acylation of papain by L-Bz-Arg-Nan is quite different and is not influenced by protonic dissociation with pKa values in the range 5-6. The earlier conclusion that the acylation of papain depends on two protonic dissociations each with pKa approx. 4 was confirmed. This argument is now more firmly based because titration with 2,2'-dipyridyl disulphide permits the loss of conformational integrity to be taken into account in the analysis of the kinetic data at very low pH. Methyl 2-pyridyl disulphide was synthesized by reaction of pyridine-2-thione with methyl methanethiolsulphonate and its pKa at I = 0.1 was determined by spectral analysis at 307 nm to be 2.8.(ABSTRACT TRUNCATED AT 400 WORDS)

Preferential resistance of dopaminergic neurons to the toxicity of glutathione depletion is independent of cellular glutathione peroxidase and is mediated by tetrahydrobiopterin.

Depletion of glutathione in the substantia nigra is one of the earliest changes observed in Parkinson's disease (PD) and could initiate dopaminergic neuronal degeneration. Nevertheless, experimental glutathione depletion does not result in preferential toxicity to dopaminergic neurons either in vivo or in vitro. Moreover, dopaminergic neurons in culture are preferentially resistant to the toxicity of glutathione depletion, possibly owing to differences in cellular glutathione peroxidase (GPx1) function. However, mesencephalic cultures from GPx1-knockout and wild-type mice were equally susceptible to the toxicity of glutathione depletion, indicating that glutathione also has GPx1-independent functions in neuronal survival. In addition, dopaminergic neurons were more resistant to the toxicity of both glutathione depletion and treatment with peroxides than nondopaminergic neurons regardless of their GPx1 status. To explain this enhanced antioxidant capacity, we hypothesized that tetrahydrobiopterin (BH(4)) may function as an antioxidant in dopaminergic neurons. In agreement, inhibition of BH(4) synthesis increased the susceptibility of dopaminergic neurons to the toxicity of glutathione depletion, whereas increasing BH(4) levels completely protected nondopaminergic neurons against it. Our results suggest that BH(4) functions as a complementary antioxidant to the glutathione/glutathione peroxidase system and that changes in BH(4) levels may contribute to the pathogenesis of PD.

Structure of chymopapain M the late-eluted chymopapain deduced by comparative modelling techniques and active-centre characteristics determined by pH-dependent kinetics of catalysis and reactions with time-dependent inhibitors: the Cys-25/His-159 ion-pair is insufficient for catalytic competence in both chymopapain M and papain.

Chymopapain M, the monothiol cysteine proteinase component of the chymopapain band eluted after chymopapains A and B in cation-exchange chromatography, was isolated from the dried latex of Carica papaya and characterized by kinetic and chromatographic analysis. This late-eluted chymopapain is probably a component of the cysteine proteinase fraction of papaya latex discovered by Schack [(1967) Compt. Rend. Trav. Lab. Carlsberg 36, 67-83], named papaya peptidase B by Lynn [(1979) Biochim. Biophys. Acta 569, 193-201] and partially characterized by Polgár [(1981) Biochim. Biophys. Acta 658, 262-269] and is the enzyme with unusual specificity characteristics (papaya proteinase IV) that Buttle, Kembhavi, Sharp, Shute, Rich and Barrett [Biochem. J. (1989) 261, 469-476] claimed to be a previously undetected cysteine proteinase eluted from a cation-exchange column near to the early-eluted chymopapains. A study of the time-dependent chromatographic consequences of thiol-dependent proteolysis of the components of papaya latex is reported. Chymopapain M was isolated by (i) affinity chromatography followed by separation from papain using cation-exchange f.p.l.c. on a Mono S HR5/5 column and (ii) cation-exchange chromatography followed by an unusual variant of covalent chromatography by thiol-disulphide interchange. The existence in chymopapain M of a nucleophilic interactive Cys/His catalytic-site system analogous to those in papain (EC 3.4.22.2) and other cysteine proteinases was deduced from the characteristics shape of the pH-second-order rate constant (k) profiles for its reactions with 2,2'-dipyridyl disulphide and ethyl 2-pyridyl disulphide. Analysis of the pH-k data for the reactions of chymopapain M with the 2-pyridyl disulphides and with 4,4'-dipyridyl disulphide permits the assignment of molecular pKa values of 3.4 and 8.7 to the formation and subsequent dehydronation of the Cys-S-/His-Im+H state of the catalytic site and reveals three other kinetically influential ionizations with pKa values 3.4, 4.3 and 5.6. The pH-dependences of kcat./Km for the hydrolysis of N-acetyl-L-Phe-Gly-4-nitroanilide at 25.0 degrees C and I0.1 M catalysed by chymopapain M and papain were determined. For both enzymes, little catalytic activity (5-7% of the maximal) develops consequent on formation of the catalytic site Cys-S-/His-Im+H ion-pair state (across pKa 3.4 for both enzymes). For papain, full expression of Kcat./Km for the uncharged substrate requires only the additional hydronic dissociation with pKa 4.2. By contrast, full expression of kcat./Km for chymopapain M requires additional hydronic dissociation with pKa values of 4.3 and 5.6.(ABSTRACT TRUNCATED AT 400 WORDS)

Identification of signalling and non-signalling binding contributions to enzyme reactivity. Alternative combinations of binding interactions provide for change in transition-state geometry in reactions of papain.

1. 2-(N'-Acetyl-L-phenylalanyl)hydroxyethyl 2'-pyridyl disulphide (compound V) was synthesized, and a study of the pH-dependence of the second-order rate constant (k) for its reaction with the catalytic-site thiol group of papain (EC 3.4.22.2) was used to evaluate the consequences for transition-state geometry of the presence of a hydrophobic occupant for the S2 subsite of the enzyme in the absence of the N-H component of the P1-P2 amide bond. 2. Comparison of the pH-dependences of K for reactions of compound (V), 2-(acetamido)ethyl 2'-pyridyl disulphide (compound I) and 2-(acetoxy)ethyl 2'-pyridyl disulphide (compound III) with the cysteine-proteinase minimal catalytic-site model, benzimidazol-2-ylmethanethiol, established the activation of all of these pyridyl disulphides by hydronation and that their reactivities are relatively insensitive to structural change in the non-pyridyl part of the molecule. The marked differences in their reactivities towards papain therefore derive from binding, either directly, or indirectly via signalling mechanisms. 3. Comparison of the kinetic data for the reaction of papain with compound (V) with those for analogous reactions with reactivity probes that provide opportunities for a variety of binding interactions in the S1-S2 intersubsite region and in the S2 subsite itself lead to the following conclusions: (a) the (Gly-66) N-H...O = C less than (P1-P2 ester) interaction of papain with compound (III) provides for better binding relative to that for a probe with a simple hydrocarbon side chain, but no signalling to the catalytic site to provide a (His-159)-ImH+-assisted transition state; (b) when this interaction is augmented either by a (P1-P2 amide) N-H...O = C less than (Asp-158) interaction (compound I) or hydrophobic P2/S2 contacts (compound V), signalling to the catalytic region occurs to provide the assisted transition state; (c) when both the P2/S2 contacts and the interaction involving Gly-66 exist, provision additionally of the (P1-P2 amide) N-H...O = C less than (Asp-158) interaction [as in 2-(N'-acetyl-L-phenylalanylamino)ethyl 2'-pyridyl disulphide] serves only to assist the binding without an additional signalling effect. 4. Such studies promise to allow binding interactions that merely locate substrates in appropriate enzyme loci to be distinguished from those that transmit signals with a chemical consequence to catalytic sites.

The interplay of electrostatic fields and binding interactions determining catalytic-site reactivity in actinidin. A possible origin of differences in the behaviour of actinidin and papain.

1. The pH-dependence of the second-order rate constant (k) for the reaction of actinidin (EC 3.4.22.14) with 2-(N'-acetyl-L-phenylalanylamino)ethyl 2'-pyridyl disulphide was determined and the contributions to k of various hydronic states were evaluated. 2. The data were used to assess the consequences for transition-state geometry of providing P2/S2 hydrophobic contacts in addition to hydrogen-bonding opportunities in the S1-S2 intersubsite region. 3. The P2/S2 contacts (a) substantially improve enzyme-ligand binding, (b) greatly enhance the contribution to reactivity of the hydronic state bounded by pKa 3 (the pKa characteristic of the formation of catalytic-site-S-/-ImH+ state) and pKa 5 (a relatively minor contributor in reactions that lack the P2/S2 contacts), such that the major rate optimum occurs at pH 4 instead of at pH 2.8-2.9, and (c) reveal the kinetic influence of a pKa approx. 6.3 not hitherto observed in reactions of actinidin. 4. Possibilities for the interplay of electrostatic effects and binding interactions in both actinidin and papain (EC 3.4.22.2) are discussed.

Substrate-derived two-protonic-state electrophiles as sensitive kinetic specificity probes for cysteine proteinases. Activation of 2-pyridyl disulphides by hydrogen-bonding.

1. 2-(N'-Acetyl-L-phenylalanylamino)ethyl 2'-pyridyl disulphide [compound (III)] and 2-(acetamido)ethyl 2'-pyridyl disulphide [compound (IV)] were synthesized by acylation of the common intermediate, 2-aminoethyl 2'-pyridyl disulphide, to provide examples of chromogenic thiol-specific substrate-derived two-protonic-state electrophilic probe reagents. These two reagents, together with n-propyl 2-pyridyl disulphide [compound (II)], provide structural variation in the non-pyridyl part of the molecule from a simple hydrocarbon side chain in compound (II) to a P1-P2 amide bond in compound (IV) and further to both a P1-P2 amide bond and a hydrophobic side chain (of phenylalanine) at P2 as a potential occupant of S2 subsites. 2. These disulphides were used as reactivity probes to investigate specificity and binding-site-catalytic-site signalling in a number of cysteine proteinases by determining (a) the reactivity at pH 6.0 at 25 degrees C at I 0.1 of compound (III) (a close analogue of a good papain substrate) towards 2-mercaptoethanol, benzimidazol-2-ylmethanethiol [compound (V), as a minimal catalytic-site model], chymopapains B1-B3, chymopapain A, papaya proteinase omega, actinidin, cathepsin B and papain, (b) the effect of changing the structure of the probe as indicated above on the reactivities of compound (V) and of the last five of these enzymes, and (c) the forms of pH-dependence of the reactivities of papain and actinidin towards compound (III). 3. The kinetic data suggest that reagents of the type investigated may be sensitive probes of molecular recognition features in this family of enzymes and are capable not only of detecting differences in binding ability of the various enzymes but also of identifying enzyme-ligand contacts that provide for binding-site-catalytic-site signalling mechanisms. 4. The particular value of this class of probe appears to derive from the possibility of activating the 2-mercaptopyridine leaving group not only by formal protonation, as was recognized previously [see Brocklehurst (1982) Methods Enzymol. 87C, 427-469], but also by hydrogen-bonding to the pyridyl nitrogen atom when the appropriate geometry in the catalytic site is provided by enzyme-ligand contacts involving the non-pyridyl part of the molecule.(ABSTRACT TRUNCATED AT 400 WORDS)

Structure-function relationships in the cysteine proteinases actinidin, papain and papaya proteinase omega. Three-dimensional structure of papaya proteinase omega deduced by knowledge-based modelling and active-centre characteristics determined by two-hydronic-state reactivity probe kinetics and kinetics of catalysis.

1. A model of the three-dimensional structure of papaya proteinase omega, the most basic cysteine proteinase component of the latex of papaya (Carica papaya), was built from its amino acid sequence and the two currently known high-resolution crystal structures of the homologous enzymes papain (EC 3.4.22.2) and actinidin (EC 3.4.22.14). The method used a knowledge-based approach incorporated in the COMPOSER suite of programs and refinement by using the interactive graphics program FRODO on an Evans and Sutherland PS 390 and by energy minimization using the GROMOS program library. 2. Functional similarities and differences between the three cysteine proteinases revealed by analysis of pH-dependent kinetics of the acylation process of the catalytic act and of the reactions of the enzyme catalytic sites with substrate-derived 2-pyridyl disulphides as two-hydronic-state reactivity probes are reported and discussed in terms of the knowledge-based model. 3. To facilitate analysis of complex pH-dependent kinetic data, a multitasking application program (SKETCHER) for parameter estimation by interactive manipulation of calculated curves and a simple method of writing down pH-dependent kinetic equations for reactions involving any number of reactive hydronic states by using information matrices were developed. 4. Papaya proteinase omega differs from the other two enzymes in the ionization characteristics of the common (Cys)-SH/(His)-Im+H catalytic-site system and of the other acid/base groups that modulate thiol reactivity towards substrate-derived inhibitors and the acylation process of the catalytic act. The most marked difference in the Cys/His system is that the pKa for the loss of the ion-pair state to form -S-/-Im is 8.1-8.3 for papaya proteinase omega, whereas it is 9.5 for both actinidin and papain. Papaya proteinase omega is similar to actinidin in that it lacks the second catalytically influential group with pKa approx. 4 present in papain and possesses a catalytically influential group with pKa 5.5-6.0. 5. Papaya proteinase omega occupies an intermediate position between actinidin and papain in the sensitivity with which hydrophobic interaction in the S2 subsite is transmitted to produce changes in transition-state geometry in the catalytic site, a fact that may be linked with differences in specificity in P2-S2 interaction exhibited by the three enzymes.(ABSTRACT TRUNCATED AT 400 WORDS)

Supracrystallographic resolution of interactions contributing to enzyme catalysis by use of natural structural variants and reactivity-probe kinetics.

1. The influence on the reactivities of the catalytic sites of papain (EC 3.4.22.2) and actinidin (3.4.22.14) of providing for interactions involving the S1-S2 intersubsite regions of the enzymes was evaluated by using as a series of thiol-specific two-hydronic-state reactivity probes: n-propyl 2-pyridyl disulphide (I) (a 'featureless' probe), 2-(acetamido)ethyl 2'-pyridyl disulphide (II) (containing a P1-P2 amide bond), 2-(acetoxy)ethyl 2'-pyridyl disulphide (III) [the ester analogue of probe (II)] and 2-carboxyethyl 2'-pyridyl disulphide N-methylamide (IV) [the retroamide analogue of probe (II)]. Syntheses of compounds (I), (III) and (IV) are reported. 2. The reactivities of the two enzymes towards the four reactivity probes (I)-(IV) and also that of papain towards 2-(N'-acetyl-L-phenylalanylamino)ethyl 2'-pyridyl disulphide (VII) (containing both a P1-P2 amide bond and an L-phenylalanyl side chain as an occupant for the S2 subsite), in up to four hydronic (previously called protonic) states, were evaluated by analysis of pH-dependent stopped-flow kinetic data (for the release of pyridine-2-thione) by using an eight-parameter rate equation [described in the Appendix: Brocklehurst & Brocklehurst (1988) Biochem. J. 256, 556-558] to provide pH-independent rate constants and macroscopic pKa values. The analysis reveals the various ways in which the two enzymes respond very differently to the binding of ligands in the S1-S2 intersubsite regions despite the virtually superimposable crystal structures in these regions of the molecules. 3. Particularly striking differences between the behaviour of papain and that of actinidin are that (a) only papain responds to the presence of a P1-P2 amide bond in the probe such that a rate maximum at pH 6-7 is produced in the pH-k profile in place of the rate minimum, (b) only in the papain reactions does the pKa value of the alkaline limb of the pH-k profile change from 9.5 to approx. 8.2 [the value characteristic of a pH-(kcat./Km) profile] when the probe contains a P1-P2 amide bond, (c) only papain reactivity is affected by two positively co-operative hydronic dissociations with pKI congruent to pKII congruent to 4 and (d) modulation of the reactivity of the common -S(-)-ImH+ catalytic-site ion-pair (Cys-25/His-159 in papain and Cys-25/His-162 in actinidin) by hydronic dissociation with pKa approx. 5 is more marked and occurs more generally in reactions of actinidin than is the case for papain reactions.(ABSTRACT TRUNCATED AT 400 WORDS)

Tetrahydrobiopterin scavenges superoxide in dopaminergic neurons.

Increased oxidative stresses are implicated in the pathogenesis of Parkinson's disease, and dopaminergic neurons may be intrinsically susceptible to oxidative damage. However, the selective presence of tetrahydrobiopterin (BH(4)) makes dopaminergic neurons more resistant to oxidative stress caused by glutathione depletion. To further investigate the mechanisms of BH(4) protection, we examined the effects of BH(4) on superoxide levels in individual living mesencephalic neurons. Dopaminergic neurons have intrinsically lower levels of superoxide than nondopaminergic neurons. In addition, inhibiting BH(4) synthesis increased superoxide in dopaminergic neurons, while BH(4) supplementation decreased superoxide in nondopaminergic cells. BH(4) is also a cofactor in catecholamine and NO production. In order to exclude the possibility that the antioxidant effects of BH(4) are mediated by dopamine and NO, we used fibroblasts in which neither catecholamine nor NO production occurs. In fibroblasts, BH(4) decreased baseline reactive oxygen species, and attenuated reactive oxygen species increase by rotenone and antimycin A. Physiologic concentrations of BH(4) directly scavenged superoxide generated by potassium superoxide in vitro. We hypothesize that BH(4) protects dopaminergic neurons from ordinary oxidative stresses generated by dopamine and its metabolites and that environmental insults or genetic defects may disrupt this intrinsic capacity of dopaminergic neurons and contribute to their degeneration in Parkinson's disease.

Consequences of molecular recognition in the S1-S2 intersubsite region of papain for catalytic-site chemistry. Change in pH-dependence characteristics and generation of an inverse solvent kinetic isotope effect by introduction of a P1-P2 amide bond into a two-protonic-state reactivity probe.

1. The pH-dependences of the second-order rate constant (k) for the reactions of papain (EC 3.4.22.2) with 2-(acetamido)ethyl 2'-pyridyl disulphide and with ethyl 2-pyridyl disulphide and of k for the reaction of benzimidazol-2-ylmethanethiol (as a minimal model of cysteine proteinase catalytic sites) with the former disulphide were determined in aqueous buffers at 25 degrees C at I 0.1. 2. Of these three pH-k profiles only that for the reaction of papain with 2-(acetamido)ethyl 2'-pyridyl disulphide has a rate maximum at pH approx. 6; the others each have a rate minimum in this pH region and a rate maximum at pH 4, which is characteristic of reactions of papain with other 2-pyridyl disulphides that do not contain a P1-P2 amide bond in the non-pyridyl part of the molecule. 3. The marked change in the form of the pH-k profile consequent upon introduction of a P1-P2 amide bond into the probe molecule for the reaction with papain but not for that with the minimal catalytic-site model is interpreted in terms of the induction by binding of the probe in the S1-S2 intersubsite region of the enzyme of a transition-state geometry in which nucleophilic attack by the -S- component of the catalytic site is assisted by association of the imidazolium ion component with the leaving group. 4. The greater definition of the rate maximum in the pH-k profile for the reaction of papain with an analogous 2-pyridyl disulphide reactivity probe containing both a P1-P2 amide bond and a potential occupant for the S2 subsite [2-(N'-acetyl-L-phenylalanylamino)ethyl 2'-pyridyl disulphide [Brocklehurst, Kowlessur, O'Driscoll, Patel, Quenby, Salih, Templeton, Thomas & Willenbrock (1987) Biochem. J. 244, 173-181]) suggests that a P2-S2 interaction substantially increases the population of transition states for the imidazolium ion-assisted reaction. 5. The overall kinetic solvent 2H-isotope effect at pL 6.0 was determined to be: for the reaction of papain with 2,2'-dipyridyl disulphide, 0.96 (i.e. no kinetic isotope effect), for its reaction with the probe containing only the P1-P2 amide bond, 0.75, for its reaction with the probe containing both the P1-P2 amide bond and the occupant for the S2 subsite, 0.61, and for kcat./Km for its catalysis of the hydrolysis of N-methoxycarbonylglycine 4-nitrophenyl ester, 0.67.(ABSTRACT TRUNCATED AT 400 WORDS)