Arginyl residues: anion recognition sites in enzymes.

Chemical modification with 2,3-butanedione in borate buffer indicates that nine of ten glycolytic enzymes studied contain arginyl residues at their active sites. Fructose-1,6-diphosphatase also has arginines at its binding site for the allosteric inhibitor, adenosine monophosphate. These and other data suggest that, as a general rule, enzymes acting on anionic substrates or cofactors will probably contain arginyl residues as components of their ligand binding sites. This could account in part for the relatively infrequent occurrence of arginine in proteins.

The enzymic nature of antibody catalysis: development of multistep kinetic processing.

Detailed kinetic investigations of a catalytic antibody that promotes the hydrolyses of an anilide and phenyl ester show that this catalyst uses a multistep kinetic sequence resembling that found in serine proteases to hydrolyze its substrates, although antibody was elicited to a single transition-state analog. Like the serine proteases the antibody catalyzes the hydrolysis reactions through a putative covalent intermediate, but unlike the enzymes it may use hydroxide ion to cleave the intermediates. Nevertheless, the antibody is a potent catalyst with turnover at higher pH values rivaling that of chymotrypsin. This analysis also reveals that turnover by the antibody is ultimately limited by product desorption, suggesting that improvements in catalytic efficiency may be achieved by judicious changes in the structure of the substrate, so that it is not superimposable on that of the eliciting hapten.

Chemical modification of histidyl and lysyl residues in yeast enolase.

Modification of yeast enolase (2-phospho-D-glycerate hydro-lyase, EC 4.2.1.11) by diethyl pyrocarbonate at either pH 6.1 or 6.6 caused a biphasic inactivation of the enzyme. In the presence of excess Mg2+, either an equilibrium mixture of substrates or 3-phosphoglycerate, a competitive inhibitor, prevented the second slower phase of inactivation, but had no effect on the first rapid phase. Complete inactivation by diethyl pyrocarbonate correlates with the modification of six histidyl residues/subunit, while 3-phosphoglycerate protects two histidyl residues/subunit from modification. Modification of enolase by two lysine-specific reagents, 2,4,6-trinitrobenzenesulfonate and pyridoxal 5'-phosphate, at pH 8.3 caused a slow loss of enzyme activity. However, substrates did not significantly protect against inactivation by either reagent, and inactivation with 2,4,6-trinitrobenzenesulfonate correlates with the modification of 18 lysyl residues/enzyme subunit.

Creatine kinase: a role for arginine-95 in creatine binding and active site organization.

Sequence homology analysis reveals that arginine-95 is fully conserved in 29 creatine kinases sequenced to date, but fully conserved as a tyrosine residue in 16 arginine kinases. Site-directed mutants of rabbit muscle creatine kinase (rmCK) were prepared in which R95 was replaced by a tyrosine (R95Y), alanine (R95A), or lysine (R95K). Kinetic analysis of phosphocreatine formation for each purified mutant showed that recombinant native rmCK and all R95 mutants follow a random-order, rapid-equilibrium mechanism. However, we observed no evidence for synergism of substrate binding by the recombinant native enzyme, as reported previously [Maggio et al., (1977) J. Biol. Chem. 252, 1202-1207] for creatine kinase isolated directly from rabbit muscle. The catalytic efficiencies of R95Y and R95A are reduced approximately 3000- and 2000-fold, respectively, compared to native enzyme, but that of R95K is reduced only 30-fold. The major contribution to the reduction of the catalytic efficiency of R95K is a 5-fold reduction in the affinity for creatine. This suggests that while a basic residue is required at position 95 for optimal activity, R95 is not absolutely essential for binding or catalysis in CK. R95Y has a significantly lower affinity for creatine than the native enzyme, but it also displays a somewhat lower affinity for MgATP and 100-fold reduction in k(cat). Interestingly, R95A appears to bind either creatine or MgATP first with affinities similar to those for the native enzyme, but it has a 10-fold lower affinity for the second substrate, suggesting that replacement of R95 by an alanine disrupts the active site organization and reduces the efficiency of formation of the catalytically competent ternary complex.

4-Hydroxy-3-nitrophenylglyoxal. A chromophoric reagent for arginyl residues in proteins.

The chromophoric reagent, 4-hydroxy-3-nitrophenylglyoxal, is highly selective for the modification of arginine in aqueous solution at pH 7--9. The reagent also inactivates creatine kinase (ATP:creatine N-phosphotransferase, EC 2.7.3.2) in a manner analogous to that reported with phenylglyoxal.

Selective phenylglyoxalation of functionally essential arginyl residues in the erythrocyte anion transport protein.

The red cell anion transport protein, band 3, can be selectively modified with phenylglyoxal, which modifies arginyl residues (arg) in proteins, usually with a phenylglyoxal: arg stoichiometry of 2:1. Indiscriminate modification of all arg in red cell membrane proteins occurred rapidly when both extra- and intracellular pH were above 10. Selective modification of extracellularly exposed arg was achieved when ghosts with a neutral or acid intracellular pH were treated with phenylglyoxal in an alkaline medium. The rate and specificity of modification depend on the extracellular chloride concentration. At 165 mM chloride maximum transport inactivation was accompanied by the binding of four phenylglyoxals per band 3 molecule. After removal of extracellular chloride, maximum transport inhibition was accompanied by the incorporation of two phenylglyoxals per band 3, which suggests that transport function is inactivated by the modification of a single arg. After cleavage of band 3 with extracellular chymotrypsin, [14C]phenylglyoxal was located almost exclusively in a 35,000-dalton peptide. In contrast, the primary covalent binding site of the isothiocyanostilbenedisulfonates is a lysyl residue in the second cleavage product, a 65,000-dalton fragment. This finding supports the view that the transport region of band 3 is composed of strands from both chymotryptic fragments. The binding of phenylglyoxal and the stilbene inhibitors interfered with each other. The rate of phenylglyoxal binding was reduced by a reversibly binding stilbenedisulfonate (DNDS), and covalent binding of [3H]DIDS to phenylglyoxal-modified membranes was strongly delayed. At DIDS concentrations below 10 10 micrometers, only 50% of the band 3 molecules were labeled with [3H]-DIDS during 90 min at 38 degrees C, thereby demonstrating an interaction between binding of the two inhibitors to the protomers of the oligomeric band 3 molecules.

Functional carboxyl groups in the red cell anion exchange protein. Modification with an impermeant carbodiimide.

Anion exchange in human red blood cell membranes was inactivated using the impermeant carbodiimide 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)-carbodiimide (EAC). The inactivation time course was biphasic: at 30 mM EAC, approximately 50% of the exchange capacity was inactivated within approximately 15 min; this was followed by a phase in which irreversible exchange inactivation was approximately 100-fold slower. The rate and extent of inactivation was enhanced in the presence of the nucleophile tyrosine ethyl ester (TEE), suggesting that the inactivation is the result of carboxyl group modification. Inactivation (to a maximum of 10% residual exchange activity) was also enhanced by the reversible inhibitor of anion exchange 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS) at concentrations that were 10(3)-10(4) times higher than those necessary for inhibition of anion exchange. The extracellular binding site for stilbenedisulfonates is essentially intact after carbodiimide modification: the irreversible inhibitor of anion exchange 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) eliminated (most of) the residual exchange activity: DNDS inhibited the residual (DIDS-sensitive) Cl- at concentrations similar to those that inhibit Cl- exchange of unmodified membranes: and Cl- efflux is activated by extracellular Cl-, with half-maximal activation at approximately 3 mM Cl-, which is similar to the value for unmodified membranes. But the residual anion exchange function after maximum inactivation is insensitive to changes of extra- and intracellular pH between pH 5 and 7. The titratable group with a pKa of approximately 5.4, which must be deprotonated for normal function of the native anion exchanger, thus appears to be lost after EAC modification.

Irreversible inactivation of red cell chloride exchange with phenylglyoxal, and arginine-specific reagent.

Chloride exchange in resealed human erythrocyte ghosts can be irreversibly inhibited with phenylglyoxal, a reagent specific for the modification of arginyl residues in proteins. Phenylglyoxal inhibits anion transport in two distinct ways. At 0 degrees C, inhibition is instantaneous and fully reversible, whereas at higher temperature in an alkaline extracellular medium, covalent binding of phenylglyoxal leads to an irreversible inhibition of the transport membranes system. Indiscriminate modification of membrane arginyl residues was prevented by reacting the with phenylglyoxal in an alkaline extracellular medium while maintaining intracellular pH near neutrality. The rate of modification of anion transport depends on phenylglyoxal concentration, pH, temperature, and the presence of anions and reversible inhibitors of the anion transport system in fashions that are fully compatible with the conclusion that phenylglyoxal modifies arginyl residues that are essential for anion binding and translocation. Phenylglyoxal reacts rapidly with the deprotonated form of the reactive groups. It is proposed that the effects of anions and of negatively charged transport inhibitors on the rate of irreversible binding of phenylglyoxal are related to the effects of the anions on a positive interfacial potential. This potential determines the local pH, and thereby the concentration of deprotonated groups, in an exofacial region of the anion transport protein.

A structural role for arginine in proteins: multiple hydrogen bonds to backbone carbonyl oxygens.

We propose that arginine side chains often play a previously unappreciated general structural role in the maintenance of tertiary structure in proteins, wherein the positively charged guanidinium group forms multiple hydrogen bonds to backbone carbonyl oxygens. Using as a criterion for a "structural" arginine one that forms 4 or more hydrogen bonds to 3 or more backbone carbonyl oxygens, we have used molecular graphics to locate arginines of interest in 4 proteins: Arg 180 in Thermus thermophilus manganese superoxide dismutase, Arg 254 in human carbonic anhydrase II, Arg 31 in Streptomyces rubiginosus xylose isomerase, and Arg 313 in Rhodospirillum rubrum ribulose-1,5-bisphosphate carboxylase/oxygenase. Arg 180 helps to mold the active site channel of superoxide dismutase, whereas in each of the other enzymes the structural arginine is buried in the "mantle" (i.e., inside, but near the surface) of the protein interior well removed from the active site, where it makes 5 hydrogen bonds to 4 backbone carbonyl oxygens. Using a more relaxed criterion of 3 or more hydrogen bonds to 2 or more backbone carbonyl oxygens, arginines that play a potentially important structural role were found in yeast enolase, Bacillus stearothermophilus glyceraldehyde-3-phosphate dehydrogenase, bacteriophage T4 and human lysozymes, Enteromorpha prolifera plastocyanin, HIV-1 protease, Trypanosoma brucei brucei and yeast triosephosphate isomerases, and Escherichia coli trp aporepressor (but not trp repressor or the trp repressor/operator complex).(ABSTRACT TRUNCATED AT 250 WORDS)

Inactivation of human thrombin by water-soluble carbodiimides. The essential carboxyl has a pKa of 5.6 and is one other than Asp-189.

Human thrombin is inactivated by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide (EAC) by a second order process. A plot of the pseudo-first order rate constant for inactivation by 20 mM EDC at different pH values from 4.2 to 7.7 at 25 degrees C shows that this inactivation is due to the modification of a protonated carboxyl with a pKa of 5.6. The rate of inactivation by EDC at pH 6.0 is reduced, but not eliminated by saturating levels of the competitive inhibitor dansyl-L-arginine N-(3-ethyl-1,5-pentanediyl)amide, suggesting that the essential carboxyl modified is one other than Asp-189 in the substrate specificity pocket of thrombin. Complete inactivation by 14C-EAC correlates with the incorporation of approximately 2.5-3 EACs per thrombin.

A comparison of the effects of cyanide, hydrogen peroxide, and phenylglyoxal on eucaryotic and procaryotic Cu,Zn superoxide dismutases.

The Cu,Zn superoxide dismutases from bovine liver, yeast, Caulobacter crescentus, and Photobacter leiognathi were compared for their susceptibilities to inhibition by cyanide and to inactivation by hydrogen peroxide and phenylglyoxal. All of these enzymes were affected by these reagents, albeit with some differences in sensitivity. The yeast and the bacterial enzymes were thus more sensitive to cyanide than was the bovine enzyme, while the bovine and the yeast enzymes were inactivated more rapidly by hydrogen peroxide and less rapidly by phenylglyoxal than were their bacterial counterparts. The qualitative similarities in the behavior of all of these enzymes suggest overriding similarities in their active site regions. However, a quantitative comparison of the data suggests that the bacterial enzymes are more like each other than they are like the eucaryotic enzymes, and furthermore, are more like the yeast enzyme than the bovine enzyme.

An essential arginyl residue at the nucleotide binding site of creatine kinase.

Treatment of rabbit muscle creatine kinase (EC 2.4.3.2) with either butanedione in borate buffer or phenylglyoxal in Veronal buffer decreases enzymatic activity correlating with the modification of a single arginyl residue per subunit of the dimeric enzyme. Very little activity is lost when modification is performed in the presence of MgATP or MgADP. Nucleotide binding to the modified enzyme is virtually abolished as determined by ultraviolet difference spectroscopy. The data suggest that an arginyl residue plays an essential role in the enzymatic mechanism of creatine kinase, probably as a recognition site for the negatively charged oligophosphate moiety of the nucleotide.

Phosphoglycerate mutase has essential arginyl residues.

Phosphoglycerate mutase is inactivated by butanedione in borate buffer. Inactivation by 0.13 mM reagent correlates with the modification of one arginyl residue per subunit, and is prevented by either 2, 3-diphosphoglycerate or 3-phosphoglycerate. With 0.50 mM butanedione, inactivation is accompanied by the modification of three arginyl residues per subunit, two of which are protected by the combined presence of cofactor and substrate.

Hydroperoxide anion, HO-2, is an affinity reagent for the inactivation of yeast Cu,Zn superoxide dismutase: modification of one histidine per subunit.

Yeast Cu,Zn superoxide dismutase is inactivated by H2O2 at alkaline pH, and complete inactivation correlates with the modification of 1.0 histidine per subunit. At elevated concentrations of H2O2, a saturation process is evident and is characterized by kmax, the maximum pseudo-first-order rate constant for inactivation, and Kinact, the total hydrogen peroxide concentration at which the enzyme is half-saturated. In the pH range from 9.0 to 11.5 at 25 degrees C, kmax remains constant at 0.54 +/- 0.03 min-1, but Kinact decreases progressively with increasing pH, from 15.5 mM at pH 9.0 to 1.11 mM at pH 11.5. It is proposed that the reason for the observed increased affinity with increasing pH is that the reactive species is not H2O2 per se, but rather the HO-2 anion (the pKa for H2O2 is 11.6). An increase in pH would thus lead to an increased concentration of HO-2 at a fixed total peroxide concentration, and saturation would occur at a lower total peroxide concentration. By analogy with other anions, it is proposed that HO-2 coordinates directly to the Cu ion to form the reactive complex. Once the enzyme-peroxide complex is formed, however, the rate-determining step leading to modification of histidine and loss of activity is independent of pH between 9.0 and 11.5.

Affinity inactivation of bovine Cu,Zn superoxide dismutase by hydroperoxide anion, HO2-.

Bovine liver Cu,Zn superoxide dismutase (SOD) is inactivated by hydrogen peroxide at alkaline pH, and full inactivation correlates with the loss of 1.1 histidine/subunit. At each pH utilized, saturation of the rate of inactivation is observed. This process is characterized by a half-saturation constant for peroxide and a maximum pseudo-first-order rate constant for inactivation. At 25 degrees C, the former decreases from 15.7 to 3.2 mM as the pH is increased from 9.0 to 11.5, while the latter increases from 0.83 to 2.43 per min over the same pH range. We have previously (Arch. Biochem. Biophys. 224, 579 (1983] proposed that the true affinity reagent for the inactivation of yeast SOD is the hydroperoxide anion, and we now believe the same is true for bovine SOD. However, a subtle difference between the two enzymes exists, for while the maximum pseudo-first-order rate constant for inactivation of bovine SOD increases with increasing pH, the same parameter for the yeast enzyme is pH-independent.

Stability of water-soluble carbodiimides in aqueous solution.

A dimethylbarbituric acid reagent has been used to follow the kinetics of loss of two water-soluble carbodiimides, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and the structurally related 1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide (EAC), in aqueous solution as a function of pH and added chemical reagents. In 50 mM 2-(N-morpholino)ethanesulfonic acid at 25 degrees C, EDC has t1/2 values of 37, 20, and 3.9 h at pH 7.0, 6.0, and 5.0, respectively, while the corresponding values for EAC are 12, 2.9, and 0.32 h. Iodide, bromide, or chloride, at 0.1 M, has very little or no effect on carbodiimide stability. However, 0.1 M glycine methyl ester or 0.1 M ethylenediamine causes a significant increase in the rate of loss of EAC and EDC, while the presence of 0.1 M phosphate, 0.1 M hydroxylamine, or 0.01 M ATP decreases the half-lives to less than or equal to 0.4 h at all pH values.

Inactivation of bovine thrombin by water-soluble carbodiimides: the essential carboxyl group has a pKa of 5.51.

Bovine thrombin is rapidly and completely (greater than 99%) inactivated by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) in a pseudo-first-order process. A plot of the pseudo-first-order rate constant for inactivation by 20 mM EDC at different pH values from pH 4.0 to 7.7 at 25 degrees C shows that inactivation is critically dependent on the protonated form of an acidic side chain with a pKa of 5.51. Significant protection against inactivation is provided by the competitive inhibitor dansyl-L-arginine N-(3-ethyl-1,5-pentanediyl)amide, suggesting that the essential carboxyl group may be involved in substrate binding. 1-Ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide (EAC) inactivates thrombin much more rapidly than EDC under the same conditions.