Two polymorphs of a covalent complex between papain and a diazomethylketone inhibitor.

The three-dimensional structure of two polymorphs of a ZLFG-CH2-papain covalent complex has been determined by X-ray crystallography. The structures indicate that: (i) the methylene carbon atom of the inhibitor is covalently bound to the Sgamma atom of Cys25 of papain; (ii) the hydrophobic S2 pocket formed by Pro68, Val133, Val157, and Asp158 is occupied by the inhibitor's phenylalanyl P2 side chain; (iii) extensive hydrogen bonding and hydrophobic interactions are responsible for the interaction of the inhibitor with the enzyme. Comparison with similar structures suggests that in covalent complexes preservation of main chain-main chain interactions between the enzyme and the inhibitor may have higher priority than the P-S interactions.     

Presence of O-linked oligosaccharide on a threonine residue in the human transferrin receptor.

We have previously demonstrated that the human transferrin receptor (TfR) of approximately 90 kDa contains Ser/Thr-linked (O-linked) oligosaccharides. In the present study, we report our identification of the site of attachment of the O-linked oligosaccharides in the receptor. A 70 kDa fragment from the external domain of the TfR was generated by trypsin treatment of the [3H]glucosamine-labelled receptor purified from human K562 cells. The beta-elimination of the intact TfR, but not the 70 kDa fragment, released Gal-[3H]Gal-NAcitol, indicating that the 70 kDa fragment lacks O-linked oligosaccharides. In the remaining 20 kDa fragment there are three potential sites (Thr96, Thr104 and Ser106) for O-glycosylation in the extracellular domain. To identify which of these residues are O-glycosylated, both the [3H]Thr- and [3H]Ser-labelled TfR were directly treated with mild base to effect beta-elimination, and the radiolabelled amino acids and their derivatives were analysed. Approximately 2% of the total radiolabelled Thr, but no radiolabelled Ser, was converted to expected beta-elimination products by this treatment. These and other results demonstrate that only one O-linked oligosaccharide is present in the TfR and that it occurs on either Thr96 or Thr104. From human serum we purified the cleaved, soluble form of the TfR (s-TfR), which contains Thr104, but lacks Thr96. The s-TfR was sensitive to O-glycanase and bound to Jacalin lectin, indicating that the s-TfR contains an O-linked oligosaccharide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mutational analysis of the three conserved valine residues of insulin and a proposal of "isosteric residue"

To further understand the role of the three conserved Val residues in insulin, B12Val, B18Val, and A3Val, five insulin mutants-[A3Ser]insulin, [B12Thr]insulin, (desB30)[B12Ser]insulin, [B18Thr] insulin, and [B18Leu]insulin--were obtained by means of site-directed mutagenesis and their receptor-binding activities as well as in vivo biological potencies were measured. The two B18 mutants, [B18Thr]insulin and [B18Leu]insulin, both retained relatively high receptor-binding activities (70% and 30% of native porcine insulin, respectively) as well as relatively high in vivo biological potencies. The receptor-binding activities of [B12Thr]insulin and (desB30)[B12Ser]insulin were 5.1% and 0.2%, respectively. However the in vivo biological potency of [B12Thr]insulin was still about 50% of native insulin, whereas that of (desB30)[B12Ser]insulin decreased drastically. The [A3Ser]insulin retained 1.4% of the receptor-binding activity and low in vivo biological potency. These results, together with previous reports showed that when the three conserved Val residues were replaced by residues containing a beta-branched side-chain, such as Thr or Ile, the insulin mutants retained higher biological activities than those mutants replaced by other residues. Here we propose that Val, Thr, and Ile are "isosteric residues' because they all contain a beta-branched side-chain. This proposal may have perhaps general significance in protein design and protein engineering.     

A single residue mutation of 5-enoylpyruvylshikimate-3-phosphate synthase in Pseudomonas stutzeri enhances resistance to the herbicide glyphosate

A novel class II 5-enoylpyruvylshikimate-3-phosphate synthase (EPSPS) was identified from Pseudomonas stutzeri A1501 by complementation of an Escherichia coli auxotrophic aroA mutant. The single amino acid substitution of serine (Ser) for asparagine (Asn)-130 of the A1501 EPSPS enhanced resistance to 200 mM glyphosate. The mutated EPSPS had a 2.5-fold increase for IC(50) [glyphosate] value, a 2-fold increase for K (i) [glyphosate] value, but a K (m) [PEP] value similar to that of wild type. The effect of the single residue mutation on glyphosate resistance was also analyzed using a computer-based three-dimensional model.     

Completion of the amino acid sequence of papain

Papain was inhibited with bromo[2-(14)C]acetic acid, the tertiary structure of the inhibited enzyme was unfolded and the disulphide bridges were reduced with mercaptoethanol and aminoethylated. Digestion with trypsin gave a radioactive peptide consisting of residues 18-58 inclusive and containing therefore the sequence of the thirteen unknown residues 29-41 in the primary sequence of papain. This peptide was digested with pepsin to give a radioactive peptide consisting of residues 18-47, which after digestion with 0.4m-hydrochloric acid gave a radioactive peptide consisting of residues 24-43 inclusive. Further digestion with 6m-hydrochloric acid gave peptides that were used to determine the sequence: Ser-Ala-Val-Val-Thr-Ile-Glx-Gly-Ile-Ile-Lys-Ile-Arg for the residues 29-41, so completing the amino acid sequence of papain.     

Location of the sites of reaction of N-ethylmaleimide in papain and chymotryptic fragments of the gizzard myosin heavy chain

The thiol of the gizzard myosin heavy chain, which reacts most rapidly with N-ethylmaleimide (MalNEt), has been located in the subfragment 2 region of myosin rod by fragmentation of [14C]-MalNEt-labeled myosin with papain and chymotrypsin. MalNEt reacts more slowly with thiols present in the 70- and 25-kilodalton (kDa) papain fragments of subfragment 1. The reaction of MalNEt with thiols present in these regions is increased on addition of ATP by factors of 2 and 10, respectively, when myosin is modified in 0.45 M NaCl where it is present in the extended, 6S conformation. The rate of increase of Mg2+-activated adenosinetriphosphatase (ATPase) activity, which reflects the loss of ability of myosin to assume the folded, 10S conformation, and the rate of loss of K+-EDTA-activated activity produced by MalNEt are both accelerated 5- to 10-fold on addition of ATP. The rates at which ATPase activities change agree closely to the reaction rates of MalNEt with the 25-kDa region of subfragment 1; therefore, the changes in these activities can be attributed to modification of a thiol of the 25-kDa segment. An increase in actin-activated ATPase activity produced by reaction of myosin with MalNEt in 0.45 M NaCl is accelerated by ATP by a factor of at least 4. Reaction with [14C]MalNEt in the presence of MgATP and 0.2 M NaCl, where myosin is in the 10S form, inhibits the incorporation of radioactive MalNEt into the 25-kDa papain fragment of subfragment 1. It also prevents the increase in actin-activated ATPase activity and preserves the ability of myosin to assume the 10S form.     

Distinct biological activities of recombinant forms of human interleukin-2 in vivo

 The biological activity of all recombinant forms of interleukin-2 (IL-2) is based upon an in vitro lymphocyte proliferation assay and measured in international units (IU). Numerous in vitro investigations have suggested that there may be different cellular effects of recombinant human IL-2 retaining the natural sequence (nIL-2) as compared to another recombinant form containing a serine substitution at amino acid position 125 ([Ser]IL-2). In the present study we investigated whether nIL-2 and [Ser]IL-2 cause similar patterns of systemic toxicities. C57BL/6 mice were treated with identical doses of either nIL-2 or [Ser]IL-2, as measured in IU, for 3 days and had blood and tissues removed for analysis of lymphocyte activation and organ dysfunction. The administration of nIL-2 had considerably greater effects on lymphocyte activation than did [Ser]IL-2, causing much greater up-regulation of the α subunit of the IL-2 receptor and the adhesion molecule lymphocyte function-associated antigen-1. Furthermore, nIL-2 induced more organ edema than did [Ser]IL-2 and caused hepatocellular injury, which was absent in mice treated with [Ser]IL-2. These data demonstrate that equivalent doses, measured in IU, of nIL-2 and [Ser]IL-2 have profoundly different effects on the induction of organ toxicity, suggesting that the IU standard may not be appropriate for the measurement of many in vivo biological activities. Received: 30 August 1996 / Accepted: 8 November 1996     

Receptor binding and biological activity of [SerB24]-insulin, an abnormal mutant insulin

[SerB24]-insulin, the second structurally abnormal mutant insulin, and [SerB25]-insulin were semisynthesized and were studied for receptor binding and biological activity. Receptor binding and biological activity determined by its ability to increase 2-deoxy-glucose uptake in rat adipocytes were 0.7-3% of native insulin for [SerB24]-insulin and 3-8% for [SerB25]-insulin. Negative cooperative effect of these analogues was also markedly decreased. Immunoreactivity of [SerB24]-insulin was decreased whereas that of [SerB25]-insulin was normal. Markedly decreased receptor binding of [SerB24]-insulin appeared to be due to substitution of hydrophobic amino acid, Phe, with a polar amino acid, Ser, at B24.     

Engineering of papain: selective alteration of substrate specificity by site-directed mutagenesis

The S2 subsite specificity of the plant protease papain has been altered to resemble that of mammalian cathepsin B by site-directed mutagenesis. On the basis of amino acid sequence alignments for papain and cathepsin B, a double mutant (Val133Ala/Ser205Glu) was produced where Val133 and Ser205 are replaced by Ala and Glu, respectively, as well as a triple mutant (Val133Ala/Val157Gly/Ser205Glu), where Val157 is also replaced by Gly. Three synthetic substrates were used for the kinetic characterization of the mutants, as well as wild-type papain and cathepsin B: CBZ-Phe-Arg-MCA, CBZ-Arg-Arg-MCA, and CBZ-Cit-Arg-MCA. The ratio of kcat/KM obtained by using CBZ-Phe-Arg-MCA as substrate over that obtained with CBZ-Arg-Arg-MCA is 8.0 for the Val133Ala/Ser205Glu variant, while the equivalent values for wild-type papain and cathepsin B are 904 and 3.6, respectively. This change in specificity has been achieved by replacing only two amino acids out of a total of 212 in papain and with little loss in overall enzyme activity. However, further replacement of Val157 by Gly as in Val133Ala/Val157Gly/Ser205Glu causes an important decrease in activity, although the enzyme still displays a cathepsin B like substrate specificity. In addition, the pH dependence of activity for the Val133Ala/Ser205Glu variant compares well with that of cathepsin B. In particular, the activity toward CBZ-Arg-Arg-MCA is modulated by a group with a pKa of 5.51, a behavior that is also encountered in the case of cathepsin B but is absent with papain.(ABSTRACT TRUNCATED AT 250 WORDS)     

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)     

Minimal side-chain protection can be a successful strategy in solid-phase peptide synthesis.

The N-terminal tyrosine residue of Met-enkephalin could be readily incorporated without protection of its phenolic hydroxylgroup. Furthermore, the HF-cleaved product contained fewer impurities than that derived from hydroxyl-protected material. Despite the presence of Tyr in the center of the chain, an LH-RH antagonist, [D-Phe2, D-Trp3, D-Phe6]-LH-RH, could also be made in normal yield by incorporation of free Boc-Tyr. Syntheses of the same model peptide without protection of the Ser residue and protection of the Arg residue as the guanidine HCl salt also gave excellent yields of analog. Finally, the LH-RH inhibitor and a highly active agonist, [D-Leu6, desGly-NH2(10)]-LH-RH ethylamide, were synthesized without protection of Tyr, Ser and Arg, which enabled free peptides to be generated directly by ammonolysis and ethylaminolysis, respectively, without HF treatment. In all examples, no evidence emerged to suggest reaction of side-chains during synthesis.     

Investigation of the catalytic site of actinidin by using benzofuroxan as a reactivity probe with selectivity for the thiolate-imidazolium ion-pair systems of cysteine proteinases. Evidence that the reaction of the ion-pair of actinidin (pKI 3.0, pKII 9.6) is modulated by the state of ionization of a group associated with a molecular pKa of 5.5.

Benzofuroxan reacts with the catalytic-site thiol group of actinidin (EC 3.4.22.14, the cysteine proteinase from Actinidia chinensis) to produce stoicheiometric amounts of the chromophoric reduction product, o-benzoquinone dioxime, and of a catalytically inactive derivative of actinidin that is devoid of thiol and that is assumed to contain, initially at least, the sulphenic acid of cysteine-25. A similar result applies also to papain (EC 3.4.22.2). The rate of o-benzoquinone dioxime formation is neither increased by inclusion of 2-mercaptoethanol or hydroxylamine in the reaction mixture nor decreased by changing the solvent from H2O to 2H2O. The change of solvent was shown to be without effect also on the rate of reaction of benzofuroxan with papain. These results suggest that the reactions of benzofuroxan with both actinidin and papain involve rate-determining attack of the catalytic-site thiol group to produce an intermediate adduct that then reacts rapidly with water to form enzyme sulphenic acid and o-benzoquinone dioxime. The pH-dependence of the second-order rate constant for the reaction of benzofuroxan with actinidin was determined in the pH range 4.3-10.2. In marked contrast with the analogous reaction of papain (reported by Shipton & Brocklehurst [(1977) Biochem. J. 167, 799-810] ) the pH-k profile for the actinidin reaction clearly contains a sigmoidal component with pKa 5.5, in which k increases with decreasing pH. These data together with the molecular pKa values for S-/ImH+ ion-pair formation and decomposition (3.0 and 9.6) suggest that the combined nucleophilic-electrophilic reactivity of the ion-pair of actinidin might be controlled by the state of ionization of another ionizing group, associated with the molecular pKa of 5.5. The pH-dependence of k for the reaction of actinidin with benzofuroxan at 25 degrees C at I 0.1 in aqueous buffers containing 6.7% (v/v) ethanol is probably adequately described by: k = k1/(1 + [H+]/KI + KII/[H+]) + k2/(1 + [H+]/KII + KIII/ [H+] + k3/(1 + [H+]/KIII) in which kI = 2.55 M -1 X s -1, k2 = 1.35 M -1, k3 = 0.93 M -1 X s -1, pKI = 3.0, pKII = 5.5 and pKIII = 9.6. By contrast, the analogous reaction of papain may be described by the same equation but with kI = 0, k2 = 2.2 M -1 X s -1, k3 = 1.3 M -1 X s -1, pKII = 3.6 and pKIII = 9.0.     

Pharmacological profile of the substituted beta-lactam L-659,286: a member of a new class of human PMN elastase inhibitors

Human polymorphonuclear leukocyte elastase (PMN elastase) is inhibited by L-659,286 (7 alpha-methoxy-8-oxo-3-[[(1,2,5,6-tetrahydro-2-methyl-5,6-dioxo-1,2,4- triaz-in-3-yl)thio]methyl]-5-thia-1-aza-6R-bicyclo[4.2.O]oct-2-ene -2- pyrrolidine carboxamide-5,-dioxide) with a Ki of 0.4 microM. This inhibition is time-dependent, rapid, and only slowly reversible, with a t1/2 of greater than 3 days at 25 degrees C. L-659,286 is also highly selective for PMN elastase, as it does not inhibit thrombin, trypsin, papain, plasmin, chymotrypsin, or cathepsin G. L-659,286 administered intratracheally inhibits lung damage caused by administration via the same route of human PMN elastase into hamsters. In marmosets, L-659,286 is cleared from blood very rapidly after an intravenous injection but is recovered in bronchoalveolar lavage fluid for several hours after intratracheal administration.     

Ser2]- and [SerP2] incretin analogs: comparison of dipeptidyl peptidase IV resistance and biological activities in vitro and in vivo

Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP; also known as gastric inhibitory polypeptide) are incretin hormones that reduce postprandial glycemic excursions via enhancing insulin release but are rapidly inactivated by enzymatic N-terminal truncation. As such, efforts have been made to improve their plasma stability by synthetic modification or by inhibition of the responsible protease, dipeptidyl peptidase (DP) IV. Here we report a parallel comparison of synthetic GIP and GLP-1 with their Ser2- and Ser(P)2-substituted analogs, examining receptor binding and activation, metabolic stability, and biological effects in vivo. Both incretins and their Ser2-substituted analogs showed similar EC50s (0.16-0.52 nm) and IC50s (4.3-8.1 nm) at their respective cloned receptors. Although both phosphoserine 2-modified (Ser(PO3H2); Ser(P)) peptides were able to stimulate maximal cAMP production and fully displace receptor-bound tracer, they showed significantly right-shifted concentration-response curves and binding affinities. Ser2-substituted analogs were moderately resistant to DP IV cleavage, whereas [Ser(P)2]GIP and [Ser(P)2] GLP-1 showed complete resistance to purified DP IV. It was shown that the Ser(P) forms were dephosphorylated in serum and thus in vivo act as precursor forms of Ser2-substituted analogs. When injected subcutaneously into conscious Wistar rats, all peptides reduced glycemic excursions (rank potency: [Ser(P)2]incretins > or = [Ser2] incretins > native hormones). Insulin determinations indicated that the reductions in postprandial glycemia were at least in part insulin-mediated. Thus it has been shown that despite having low in vitro bioactivity using receptor-transfected cells, in vivo potency of [Ser(P)2] incretins was comparable with or greater than that of native or [Ser2]peptides. Hence, Ser(P)2-modified incretins present as novel glucose-lowering agents.     

Degradation of growth hormone releasing factor analogs in neutral aqueous solution is related to deamidation of asparagine residues. Replacement of asparagine residues by serine stabilizes

The incubation of a solution of the human growth hormone releasing factor analog, [Leu27] hGRF(1-32)NH2 at pH 7.4 and 37 degrees, resulted in extensive degradation of the sample. The major degradation products were identified as the peptides [beta-Asp8, Leu27] hGRF(1-32)NH2 and [alpha-Asp8, Leu27] hGRF(1-32)NH2, produced by deamidation of the Asn8 residue. When tested as growth hormone (GH) secretagogues in cultured bovine anterior pituitary cells, [beta-Asp8, Leu27] hGRF(1-32)NH2 was estimated to be 400-500 times less potent than the parent Asn8 peptide, while [alpha-Asp8, Leu27] hGRF(1-32)NH2 was calculated to be 25 times less potent than the parent Asn8 peptide. Three additional analogs of [Leu27] hGRF(1-32)NH2 containing either Ser or Asn at positions 8 and 28 were prepared and evaluated for their GH releasing activity and stability in aqueous phosphate buffer (pH 7.4, 37 degrees). Based on disappearance kinetics, [Leu27] hGRF(1-32)NH2 had a half-life of 202 h while the other analogs had the following half-lives: [Leu27, Asn28] hGRF(1-32)NH2 (150 h); [Ser8, Leu27, Asn28] hGRF(1-32)NH2 (746 h); and [Ser8, Leu27] hGRF(1-32)NH2 (1550 h). After 14 days, incubated samples of the Asn8 analogs lost GH releasing potency, while the Ser8 analogs retained full potency. The potential for loss of biological activity brought about by deamidation of other engineered peptides and proteins should be considered in their design.     

Structural and functional characterization of transmembrane segment IX of the NHE1 isoform of the Na+/H+ exchanger

The Na(+)/H(+) exchanger isoform 1 (NHE1) is an integral membrane protein that regulates intracellular pH by removing one intracellular H(+) in exchange for one extracellular Na(+). It has a large N-terminal membrane domain of 12 transmembrane segments and an intracellular C-terminal regulatory domain. We characterized the cysteine accessibility of amino acids of the putative transmembrane segment IX (residues 339-363). Each residue was mutated to cysteine in a functional cysteineless NHE1 protein. Of 25 amino acids mutated, 5 were inactive or nearly so after mutation to cysteine. Several of these showed aberrant targeting to the plasma membrane and reduced expression of the intact protein, whereas others were expressed and targeted correctly but had defective NHE1 function. Of the active mutants, Glu(346) and Ser(351) were inhibited >70% by positively charged [2-(trimethylammonium)-ethyl]methanethiosulfonate but not by anionic [2-sulfonatoethyl]methanethiosulfonate, suggesting that they are pore lining and make up part of the cation conduction pathway. Both mutants also had decreased affinity for Na(+) and decreased activation by intracellular protons. The structure of a peptide representing amino acids 338-365 was determined by using high resolution NMR in dodecylphosphocholine micelles. The structure contained two helical regions (amino acids Met(340)-Ser(344) and Ile(353)-Ser(359)) kinked with a large bend angle around a pivot point at amino acid Ser(351). The results suggest that transmembrane IX is critical with pore-lining residues and a kink at the functionally important residue Ser(351).     

Mutants of human insulin-like growth factor I with reduced affinity for the type 1 insulin-like growth factor receptor

Four mutants of human insulin-like growth factor I (hIGF I) have been purified from the conditioned media of yeast transformed with an expression vector containing a synthetic gene for hIGF I altered by site-directed mutagenesis. hIGF I has the sequence Phe-23-Tyr-24-Phe-25 which is homologous to a region in the B-chain of insulin. [Phe23,Phe24,Tyr25]IGF I, in which the sequence is altered to exactly correspond to the homologous sequence in insulin, is equipotent to hIGF I at the types 1 and 2 IGF and insulin receptors. [Leu24]IGF I and [Ser24]IGF I have 32- and 16-fold less affinity than hIGF I at the human placental type 1 IGF receptor, respectively. These peptides are 10- and 2-fold less potent at the placental insulin receptor, respectively. [Leu24]IGF I and [Ser24]IGF I have similarly reduced affinities for the type 1 IGF receptor of rat A10 and mouse L cells. Thus, the importance of the interaction of residue 24 with the receptor is conserved in several species. In three cell-based assays, [Leu24]IGF I and [Ser24]IGF I are full agonists with reduced efficacy compared to hIGF I. Desoctapeptide [Leu24]IGF I, in which the loss of aromaticity at position 24 is combined with the deletion of the carboxyl-terminal D region of hIGF I, has 3-fold lower affinity than [Leu24]IGF I for the type 1 receptor and 2-fold higher affinity for the insulin receptor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation of glutaraldehyde-crosslinked chymotrypsinogen-A. Enzymatic activity and circular dichroism studies

Phenylhydrazine does not inactivate papain or glyceraldehyde-3-phosphate dehydrogenase under anaerobic conditions. The inactivation of papain and glyceralde-hyde-3-phosphate dehydrogenase under aerobic conditions is ascribed to the oxidation of phenylhydrazine by O₂ which generates phenyldiimide and H₂O₂, both of which react with the essential sulfhydryl groups and inactivate the enzymes. Phenyldiimide generated from methyl phenylazoformate inactivates both of the sulfhydryl enzymes under anaerobic conditions. The inactivation of papain and GPD with aerobic, aqueous solutions of [¹⁴C]phenylhydrazine introduces a small amount of radioactivity into the enzymes which is discharged by dithiothreitol. The amount of radioactivity bound to papain is increased when cyanide is present in the inactivation mixture.When the β-[¹⁴C]thiocyanoalanine derivative of papain is treated with phenylhydrazine the radioactivity is discharged from the enzyme. Cyanide evidently reacts with the sulfenic acid derivative of papain to form a thiocyanate derivative. Phenylhydrazine presumably displaces cyanide from the thiocyanate derivative to form a sulfenyl hydrazide derivative to account for the increased incorporation of [¹⁴C]phenylhydrazine when papain is inactivated with aerobic solutions of [¹⁴C]-phenylhydrazine in the presence of cyanide. When the sulfhydryl group of papain is oxidized to a sulfenic acid with H₂O₂ and then treated with [¹⁴C]phenylhydrazine, ¹⁴C is not incorporated into the enzyme. These experiments suggest that the H₂O₂ in the aerobic solutions of phenylhydrazine oxidizes the sulfhydryl group at the active site of papain to a sulfenic acid. The [¹⁴C]phenyldiimide in these solutions reacts to some extent with the active sulfhydryl group to form a sulfenyl hydrazide derivative.     

Identification of a selenocysteyl-tRNA(Ser) in mammalian cells that recognizes the nonsense codon, UGA

The presence of a unique opal suppressor seryl-tRNA in higher vertebrates which is converted to phosphoseryl-tRNA has been known for several years, but its function has been uncertain (see Hatfield, D. (1985) Trends Biochem. Sci. 10, 201-204 for review). In the present study, we demonstrate that this tRNA species also occurs in vivo as selenocysteyl-tRNA(Ser) suggesting that it functions both as a carrier molecule upon which selenocysteine is synthesized and as a direct selenocysteine donor to a growing polypeptide chain in response to specific UGA codons. [75Se]Seleno[3H]cysteyl-tRNA(Ser) formed by administering 75Se and [3H]serine to rat mammary tumor cells (TMT-081-MS) in culture was isolated from the cell extract. The amino acid attached to the tRNA was identified as selenocysteine following its deacylation and reaction with iodoacetate and 3-bromopropionate. The resulting alkyl derivatives co-chromatographed on an amino acid analyzer with authentic carboxymethylselenocysteine and carboxyethylselenocysteine. Seryl-tRNA(Ser) and phosphoseryl-tRNA(Ser) (Hatfield, D., Diamond, A., and Dudock, B. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 6215-6219), which co-migrate on a reverse phase chromatographic column with selenocysteyl-tRNA(Ser), were also identified in extracts of TMT-018-MS cells. Hence, we propose that a metabolic pathway for selenocysteine synthesis in mammalian cells is the conversion of seryl-tRNA(Ser) via phosphoseryl-tRNA(Ser) to selenocysteyl-tRNA(Ser). In a ribosomal binding assay selenocysteyl-tRNA(Ser) recognizes UGA but not any of the serine codons. Selenocysteyl-tRNA(Ser) is deacylated more readily than seryl-tRNA(Ser) (i.e. 58% deacylation during 15 min at pH 8.0 and 37 degrees C as compared to 41%).     

Inactivation of DNase by 2-nitro-5-thiocyanobenzoic acid. II. Serine and threonine are the sites of reaction on the DNase molecule.

The amino acid compositions of various fragments isolated from DNase treated with 2-nitro-5-thiocyanobenzoic acid (NTCB) show peptide bond cleavages to be at Thr14, Ser40, and Ser135. Isolation and characterization of radioactive tryptic and chymotryptic peptides of [14C]cyano-DNase reveal four points of peptide bond cleavage; in addition to Thr14, Ser40, and Ser135, cleavage occurs at the amino end of Ser72. Approximately 2.8 mol of [14C]cyano group are incorporated in the completely inactivated enzyme, in which 0.6 residue of Thr14, 0.8 of Ser40, and approximately 0.3 each of Ser72 and Ser135 are modified. The inactivation by NTCB can also be obtained by reacting the enzyme with a mixture of 5,5'-dithiobis(2-nitrobenzoic acid), KCN, and iodoacetate which generates NTCB. The mixture facilitates the uses of K[14C]N, which is readily incorporated into the enzyme as the [14C]cyano derivative. The reaction of NTCB with serine or threonine resembles that with cysteine.