Ethoxyformylation of bovine growth hormone

Reactivity of histidines in bovine growth hormone towards ethoxyformic anhydride was investigated and localization in the molecule of two kinetically distinguishable classes was achieved, a slow class including only histidine residue 169 (k = 0.180 min-1) and a fast one composed of histidines 19 and 21 (k = 0.900 min-1). Total ethoxyformylation of bovine growth hormone brought about a complete loss of its capacity to compete with 125I-labelled hormone for rat-liver binding sites, but modification of approximately half of the fast histidine group was enough to produce an important decrease in this capacity. Circular dichroism studies indicated no significant changes in protein conformation with all three histidine residues modified. Practically full binding capacity was restored when these residues were regenerated by treatment with hydroxylamine. These results suggest that one or both of the fast reacting histidine residues are involved in bovine growth hormone binding to its specific receptors.     

Role of histidine residues in the binding mechanism of the growth hormone family

1. Reactivity of the hGH histidine residues were studied by reaction with ethoxyformic anhydride. Localization in the molecule of three kinetically distinguishable classes, each including only one residue, was achieved. 2. The first was composed of residue 151, with an apparent velocity constant k = 0.735/min, (similar to that of histidines 19 and 21 in bGH and eGH). The second histidine, 18, with a velocity constant k = 0.135/min, (similar to that of histidine 169 in the above hormones), and a third, histidine 21, which does not react at all. 3. Neither histidine 151 nor 18 seem to be involved, at least not directly, in bGH binding to specific rat liver sites, since the decrease in this capacity was only 47% after modification of the former by 77 and 65% after total modification of the latter. 4. These results, and those previously obtained with bGH and eGH, suggest that either histidine 21 is the only indispensable histidine for the binding of growth hormones to specific rat liver sites, or that histidine 21 and/or 18 (19 in bGH and eGH), are located within the growth hormone binding site interaction area.     

Reaction of the histidines of prolactin with ethoxyformic anhydride. A binding site modification.

The seven histidines of bovine prolactin were modified with ethoxyformic anhydride and two classes of reactivity were apparent: 5 histidines were in the more reactive class (k = 0.097 min-1) and 2 histidines were less reactive (k = 0.011 min-1). The activity of the modified prolactins was determined by measuring their ability to bind to prolactin receptors from rabbit mammary glands. This assay showed that prolactin was fully active when 0 to 5 histidines were modified. If all 7 residues were modified, the hormone was unable to bind to its receptor. Circular dichroism studies indicated no significant differences in conformation for prolactins which had 2 to 7 histidines modified. Modification of human growth hormone and human placental lactogen with ethoxyformic anhydride resulted in a loss of the ability of these lactogenic hormones to bind to the prolactin receptor. For all three hormones, essentially full activity was recovered when the modifying group was removed by treatment with hydroxylamine. Sequence comparisons indicate that only 2 of the 3 growth hormone histidines and 2 of 7 placental lactogen histidines were homologous with histidines in bovine prolactin and that, in each case, they correspond to His-27 and His-30 in bovine prolactin. It is postulated that these residues serve to identify a portion of the binding domain of bovine prolactin.     

The influence of uncoordinated histidines on iron release from transferrin. A chemical modification study

Histidine residues that influence the chelate-mediated removal of iron from transferrin have been investigated. Diferric human serum transferrin was chemically modified to various extents using ethoxyformic anhydride, a reagent for histidines. A kinetic analysis of the modification reaction revealed the presence of a fast reacting pool of 9 +/- .8 histidine residues and a slow reacting pool of 5.8 +/- .6 residues. There are 18 histidine residues in transferrin. The rates of modification of the two pools differed by a factor of 5. The pyrophosphate-mediated removal of iron from the two binding sites of native and partially modified transferrins was studied at pH 6.9 using desferrioximine B as a terminal iron acceptor. Under these conditions, the rate of iron removal from the NH2-terminal site was about six times faster than from the COOH-terminal site. Both rates were significantly reduced, i.e. by a factor of approximately 6-8, upon complete ethoxyformylation of all reactive histidines on the protein. The kinetic data of partially modified transferrins were analyzed by the Tsou Chen-Lu statistical method; the results are consistent with the hypothesis that modification of a single uncoordinated histidine in each of the two iron binding domains stabilizes the protein kinetically against loss of iron. The dependence of the iron removal reaction on pH is consistent with such an interpretation. The putative histidines, although not ligands, may be close to the metal in both binding sites, thus influencing the rate of iron removal by pyrophosphate. These histidines belong to the pool of rapidly modified residues and thus are readily accessible to solvent and chelators.     

Contribution of individual histidines to the global stability of human prolactin

A member of the family of hematopoietic cytokines human prolactin (hPRL) is a 23k kDa polypeptide hormone, which displays pH dependence in its structural and functional properties. The binding affinity of hPRL for the extracellular domain of its receptor decreases 500‐fold over the relatively narrow, physiologic pH range from 8 to 6; whereas, the affinity of human growth hormone (hGH), its closest evolutionary cousin, does not. Similarly, the structural stability of hPRL decreases from 7.6 to 5.6 kcal/mol from pH 8 to 6, respectively, whereas the stability of hGH is slightly increased over this same pH range. hPRL contains nine histidines, compared with hGH's three, and they are likely responsible for hPRL's pH‐dependent behavior. We have systematically mutated each of hPRL's histidines to alanine and measured the effect on pH‐dependent global stability. Surprisingly, a vast majority of these mutations stabilize the native protein, by as much as 2–3 kcal/mol. Changes in the overall pH dependence to hPRL global stability can be rationalized according to the predominant structural interactions of individual histidines in the hPRL tertiary structure. Using double mutant cycles, we detect large interaction free energies within a cluster of nearby histidines, which are both stabilizing and destabilizing to the native state. Finally, by comparing the structural locations of hPRL's nine histidines with their homologous residues in hGH, we speculate on the evolutionary role of replacing structurally stabilizing residues with histidine to introduce pH dependence to cytokine function.     

Proton nuclear magnetic resonance studies on bovine lutropin, its subunits, and on the alpha subunit of pregnant mare serum gonadotropin. Assignment of histidine resonances in the alpha subunit.

The pK values of the 3 histidine residues in the common alpha subunits of bovine and equine glycoprotein hormones have been determined from titration curves generated from their C-2 proton nuclear magnetic resonances at different pH values. Assignment of resonances to specific histidines is based on a comparison between the two species, which have 1 histidine residue in different positions in their sequences, and of the bovine alpha subunit after removal of its histidine 94 by treatment with carboxypeptidases. In both species, those histidines closest to the COOH terminus titrate with near normal pK values of 6.2. The histidine residue found in the bovine subunit at position 87 titrates with an approximate pK value of 5.4. Histidine 83, adjacent to an oligosaccharide moiety in both species, does not titrate over a pH range of 4.0 to 8.0 and thus appears inaccessible to solvent. Similarly, in bovine lutropin-beta, 1 of 3 histidine residues does not titrate between pH 5.0 and 7.0. In the intact hormone, 2 "nontitratable" histidine residues are found. Changes in the characteristics of the signals, however, preclude unambiguous assignment of these two resonances to the nontitrating histidines in the isolated subunits. It appears that changes in the environment of at least some histidines occur when the subunits combine to yield intact hormone.     

Regulation of cAMP-dissociation kinetics in lactating rat mammary gland

The cAMP-dissociation kinetics of rat mammary gland cytosols are dependent upon the temperature of cAMP association. Dissociation rates (measured at pH 6.5, 24 degrees C) were biphasic (k = 0.08-0.23 min-1 and k = 0.02 min-1) and monophasic (k-1 = 0.02 min-1) after 0 degrees C and 24 degrees C association, respectively. The temperature-dependent change from an initial fast rate to an initial slow rate was observed at all concentrations of cAMP tested from 1 to 1000 nM. When the slow-dissociating site was associated with non-radioactive 8-bromo-cAMP, the dissociation rates of [3H]-cAMP from the remaining dissociating site was slow (k = 0.02 min-1) and fast (k = 0.05 min-1) at 24 degrees C and 0 degrees C associating rate can be converted to the slow-dissociating rate by warming. When 0.2 M sodium thiocyanate was added to the association mixture at 24 degrees C, biphasic dissociation rates of k = 0.23 min-1 and k = 0.02 min-1 were observed, suggesting that the chaotropic salt blocks the interconversion of rates. The data are consistent with the model for cAMP-dependent protein kinase which exhibits two binding sites with different affinities. The type II enzyme from mammary gland cytosol exhibits in addition the phenomenon of temperature-dependent interconversion of the two binding affinities.     

Role of histidines in the binding of violaxanthin de-epoxidase to the thylakoid membrane as studied by site-directed mutagenesis

Regulation of violaxanthin de-epoxidase (VDE) involves a conformational change at low lumenal pH, followed by binding of the enzyme to the thylakoid membrane. The role of histidine residues in this process was studied by release of unbound enzyme from thylakoids upon sonication, on a pH scale from 4.7 to 7.1. The co-operativity for binding of spinach VDE (four histidines) to the membrane was found to be 3.8, with respect to protons, and had an inflexion point at pH 6.6, whereas VDE from wheat (three histidines) showed a co-operativity of 2.9 and had an inflexion point at pH 6.2. Mutant forms of VDE were constructed and probed for their binding to the outside of thylakoid membranes. With one or two histidines substituted for alanine or arginine, a lower co-operativity (1.6–2.3) was found, compared with the wild type. Based on these findings, and that the pKa value for histidine is within the range where the VDE binding takes place, we propose that protonation of the histidine residues at low pH induces the conformational change of VDE, and hence indirectly regulates binding of the enzyme to the thylakoid membrane.     

Oligonucleotide site directed mutagenesis of all histidine residues within the T4 endonuclease V gene: role in enzyme-nontarget DNA binding

In order to evaluate the contributions that histidine residues might play both in the catalytic activities of endonuclease V and in binding to nontarget DNA, the technique of oligonucleotide site directed mutagenesis was used to create mutations at each of the four histidine residues in the endonuclease V gene. Although none of the histidines were shown to be absolutely required for the pyrimidine dimer specific DNA glycosylase activity or the apurinic lyase activity, conservative amino acid changes at His16 produced enzymes with little or no catalytic activity. In addition, the evaluation of conservative and radical amino acid substitutions at positions 34, 56, and 107 is consistent with the interpretation that each of these histidines may be involved in nontarget DNA binding. The data supporting this conclusion are that histidine changes to lysine at positions 34 and 107 enhance the nontarget DNA binding activity of the mutant enzymes while neutralization of charge at His56 reduces nontarget DNA binding.     

Studies of the histidine residues of human and bovine glycoprotein hormones by nuclear magnetic resonance

Titration curves of the histidine residues in lutropin, thyrotropin, follitropin and chorionic gonadotropin have been assigned using imidazole C-2 proton nuclear magnetic resonance spectra and their estimated pK values determined. Spectra of reassociated hormone preparations, in which one or the other of their two subunits (alpha or beta) have had their accessible histidines exchanged with deuterium, permitted assignment of C-2 resonance to specific residues. Similar titration curves were found for residues which are conserved from one hormone to another. However, these conserved histidines do not have identical pK values, indicating that differences in the conformation or microenvironment around these residues occur in these hormones. Changes in some pK values also occur as a function of subunit association. The most dramatic change seen in all cases is the exposure to solvent of histidine alpha-83; in isolated alpha subunits this residue is unavailable for titration over a wide pH range. This change appears to be a general consequence of the association of the two subunits in any of these hormones. The data show that all histidines in the intact hormones are accessible to the environment, including those proposed to be in domains involved in subunit-subunit interaction.     

Inactivation of Pseudomonas iron-superoxide dismutase by hydrogen peroxide

Pseudomonas Fe-superoxide dismutase (superoxide:superoxide oxidoreductase, EC 1.15.1.1) is inactivated by hydrogen peroxide by a mechanism which exhibits saturation kinetics. The pseudo-first-order rate constant of the inactivation increased with increasing pH, with an inflection point around pH 8.5. Two parameters of the inactivation were measured in the pH range 7.8 to 9.0; the total H2O2 concentration at which the enzyme is half-saturated (K inact) was found to be independent of pH (30 mM) and the maximum rate constant for inactivation (k max) increased progressively with increasing pH, from 3.3 min-1 at pH 7.8 to 21 min-1 at pH 9.0. This evidence suggests the presence of an ionization group (pKa approximately 8.5) which does not participate in the binding of H2O2 but which affects the maximum inactivation rate of the enzyme. The loss of dismutase activity of the Fe-superoxide dismutase is accompanied by a modification of 1.6, 1.1 and 0.9 residues of tryptophan, histidine and cysteine, respectively. Since the amino acid residues of the Cr-substituted enzyme, which has no enzymatic activity, were not modified by H2O2, the active iron of the enzyme is essential for the modification of the amino acid residues.     

Identification of essential histidines in cyclodextrin glycosyltransferase isoform 1 from Paenibacillus sp. A11

The isoform 1 of cyclodextrin glycosyltransferase (CGTase, EC 2.4.1.19) from Paenibacillus sp. A11 was purified by a preparative gel electrophoresis. The importance of histidine, tryptophan, tyrosine, and carboxylic amino acids for isoform 1 activity is suggested by the modification of the isoform 1 with various group-specific reagents. Activity loss, when incubated with diethylpyrocarbonate (DEP), a histidine modifying reagent, could be protected by adding 25 mM methyl-beta-cyclodextrin substrate prior to the modification. Inactivation kinetics of isoform 1 with DEP resulted in second-order rate constants (k(inactivation)) of 29.5 M(-1)s(-1). The specificity of the DEP-modified reaction for the histidine residue was shown by the correlation between the loss of isoform activity and the increase in the absorbance at 246 nm of N-carbethoxyhistidine. The number of histidines that were modified by DEP in the absence and presence of a protective substrate was estimated from the increase in the absorbance using a specific extinction coefficient of N-carbethoxyhistidine of 3,200 M(-1)cm(-1). It was discovered that methyl-beta-CD protected per mole of isoform 1, two histidine residues from the modification by DEP. To localize essential histidines, the native, the DEP-modified, and the protected forms of isoform 1 were digested by trypsin. The resulting peptides were separated by HPLC. The peptides of interest were those with R(t) 11.34 and 40.93 min. The molecular masses of the two peptides were 5,732 and 2,540 daltons, respectively. When the data from the peptide analysis were checked with the sequence of CGTase, then His-140 and His-327 were identified as essential histidines in the active site of isoform 1.     

Nuclear magnetic resonance titration curves of histidine ring protons. A direct assignment of the resonances of the active site histidine residues of ribonuclease.

One of the four titrating histidine ring C-2 proton resonances of bovine pancreatic ribonuclease has been assigned to histidine residue 12. This was accomplished by a direct comparison of the rate of tritium incorporation into position C-2 of histidine 12 of S-peptide (residues 1 to 20) derived from ribonuclease S, with the rates of deuterium exchange of the four histidine C-2 proton resonances of ribonuclease S under the same experimental conditions. The same assignment was obtained by a comparison of the NMR titration curves of ribonuclease S, the noncovalent complex of S-peptide and S-protein (residues 21 to 124) with the results for the recombined complex in which position C-2 of histidine 12 was fully deuterated. The second active site histidine resonance was assigned to histidine residue 119 by consideration of the NMR titration results fro carboxymethylated histidines and 1-carboxymethylhistidine 119 ribonuclease. This assignment is a reversal of that originally reported, and has important implications for the interpretation of NMR titration data of ribonuclease.     

Chemical modification ofStaphylococcus aureus α-toxin by diethylpyrocarbonate: Role of histidines in its membrane-damaging properties

Summary Staphylococcus aureus -toxin causes cell damage by forming an amphiphilic hexamer that inserts into the cell membrane and generates a hydrophilic pore. To investigate the role of the three histidine residues of this toxin we modified them with diethylpyrocarbonate, obtaining N-carbethoxy-histidine whose appearance may be followed spectrophotometrically. Despite the statistical nature of random chemical modification, it was possible to establish that modification of any one of the three histidines was enough to impair -toxin activity on red blood cells and platelets. Two out of three histidines were essential for the interaction of the toxin with model membranes such as lipid vesicles and planar bilayers. Loss of lytic activity in both natural and model membranes was due both to defective binding and to defective oligomerization. When -toxin hexamers inserted into lipid vesicles were assayed for chemical modifiability two histidines per monomer were found to be protected from diethylpyrocarbonate modification, whereas only one was protected after delipidation of the oligomer with a detergent. A possible model for the role of each histidine in the monomer is presented.     

Diagnostic test for ruthenium and platinum modification of histidine residues on metalloproteins using diethylpyrocarbonate (DEPC)

The reaction of diethylpyrocarbonate (DEPC) with uncoordinated surface histidine residues on metalloproteins at pH 6.5−7.0 can be readily monitored (∼30 min) by spectrophotometric changes at ∼238 nm (ϵ = 2750 M⁻¹). No reaction is observed if prior modification of histidine by attachment of Ru(II) or Pt(II) has been carried out. In the studies with azurin and cytochrome c only one of two histidines readily undergoes DEPC modification, and likewise only one histidine is Ru modified. Thus the reaction can be used as a quick test for histidine availability, and subsequently as a test as to whether modification has been achieved. The specificity of Pt complexes for histidines is less than that of Ru. In a wider context of histidine availability only one of three histidines in Rieske's protein (N. crassa) is DEPC modified.     

Kinetics of carboxymethylation of histidine hydantoin.

The reaction of the imidazole group of histidine hydantoin with bromoacetate was studied as a model for carboxymethylation of histidine residues in proteins. pK values of 6.4 and 9.1 (25 degrees C) and apparent heats of ionization of 7.8 and 8.7 kcal/mol were determined for the imidazole and hydantoin rings, respectively. At pH values corresponding to the isoelectric points for histidine hydantoin, the rates of carboxymethylation at 12, 25, 37, and 50 degrees C were determined; the modified hydantoins were hydrolyzed to the corresponding histidine derivatives for quantitative amino acid analysis. At pH 7.72 and 25 degrees C, the imidazole tele-N was alkylated (k = 3.9 X 10(-5) M-1 s-1) twice as fast as the pros-N. The monocarboxymethyl derivatives were carboxymethylated at the same rate at the pros-N (k = 2.1 X 10(-5) M-1 s-1) but 3 times faster at the tele-N (k = 11 X 10(-5) M-1 s-1). The enthalpies of activation determined for carboxymethylation of the imidazole ring and its monocarboxymethyl derivatives were similar (15.9 +/- 0.7 kcal/mol). delta S for the four carboxymethylations was -25 +/- 2 eu. The electrostatic component of delta S (delta S es) was calculated from the influence of the dielectric constant on the reaction rate at 25 degrees C. delta S es was slightly negative (-4 +/- 1 eu) for mono- or dicarboxymethylations, indicating some charge separation in the transition state. The nonelectrostatic entropy of activation was -21 +/- 2 eu for all four carboxymethylations.     

Histidine 295 and histidine 510 are crucial for the enzymatic degradation of heparan sulfate by heparinase III

The heparinases from Flavobacterium heparinum are powerful tools in understanding how heparin-like glycosaminoglycans function biologically. Heparinase III is the unique member of the heparinase family of heparin-degrading lyases that recognizes the ubiquitous cell-surface heparan sulfate proteoglycans as its primary substrate. Given that both heparinase I and heparinase II contain catalytically critical histidines, we examined the role of histidine in heparinase III. Through a series of diethyl pyrocarbonate modification experiments, it was found that surface-exposed histidines are modified in a concentration-dependent fashion and that this modification results in inactivation of the enzyme (k(inact) = 0.20 +/- 0.04 min(-)(1) mM(-)(1)). The DEPC modification was pH dependent and reversible by hydroxylamine, indicating that histidines are the sole residue being modified. As previously observed for heparinases I and II, substrate protection experiments slowed the inactivation kinetics, suggesting that the modified residue(s) was (were) in or proximal to the active site of the enzyme. Proteolytic mapping experiments, taken together with site-directed mutagenesis studies, confirm the chemical modification experiments and point to two histidines, histidine 295 and histidine 510, as being essential for heparinase III enzymatic activity.     

Identification of the essential histidine residue at the active site of Escherichia coli dehydroquinase.

The shikimate pathway enzyme 3-dehydroquinase is very susceptible to inactivation by the group-specific reagent diethyl pyrocarbonate (DEP). Inactivation follows pseudo first-order kinetics and exhibits a second-order rate constant of 148.5 M-1 min-1. An equilibrium mixture of substrate and product substantially protects against inactivation by DEP, suggesting that residues within the active site are being modified. Complete inactivation of the enzyme correlates with the modification of 6 histidine residues/subunit as determined by difference spectroscopy at 240 nm. Enzymic activity can be restored by hydroxylamine treatment, which is also consistent with the modification occurring at histidine residues. Using the kinetic method of Tsou (Tsou, C.-L. (1962) Sci. Sin. 11, 1535-1558), it was shown that modification of a single histidine residue leads to inactivation. Ligand protection experiments also indicated that 1 histidine residue was protected from DEP modification. pH studies show that the pKa for this inactivation is 6.18, which is identical to the single pKa determined from the pH/log Vmax profile for the enzyme. A single active site peptide was identified by differential peptide mapping in the presence and absence of ligand. This peptide was found to comprise residues 141-158; of the 2 histidines in this peptide (His-143 and His-146), only one, His-143, is conserved among all type I dehydroquinases. We propose that His-143 is the active site histidine responsible for DEP-mediated inactivation of dehydroquinase and is a good candidate for the general base that has been postulated to participate in the mechanism of this enzyme.     

The reaction of the histidine residues of luteinizing hormone with ethoxyformyl anhydride.

Bovine and porcine luteinizing hormones (B-LH, P-LH) and their subunits were treated by ethoxyformyl anhydride. The acylation of the histidine residues was followed by examination of the absorbance spectrum. All the histidine residues of the luteinizing hormone molecule can be modified at pH5. However 2 His in B-LH and 1 in P-LH appear to be much less reactive at pH 5 than the others and their acylated imidazols more labile at the same pH. At neutral pH, 2 histidines in B-LH (and 1 in P-LH) become unreactive. In the case of the subunits, 1 histidine becomes unreactive in each subunit at neutral pH. These unreactive histidine residues at neutral pH are probably those which appear to be poorly reactive at pH 5. Comparison of the results obtained with B-LH and P-LH suggests that of the 2 histidine residues present in B-LH and absent in P-LH (beta 60, beta 112), only one exhibits a low reactivity. Acylation of 4 His in B-LH do not cause dissociation into subunits of the molecule but supress 95 per cent of the biological activity.