Studies on the biotin-binding site of avidin. Lysine residues involved in the active site.

Egg-white avidin was treated with 1-fluoro-2,4-dinitrobenzene. Modification of an average of one lysine residue per avidin subunit caused the complete loss of biotin binding. Tryptic peptides obtained from the 2,4-dinitrophenylated avidin were fractionated by reversed-phase h.p.l.c. Three peptides contained the 2,4-dinitrophenyl group. Amino acid analysis revealed that lysine residues 45, 94 and 111 are modified and probably comprise part of the biotin-binding site.     

Studies on the biotin-binding sites of avidin and streptavidin. Tyrosine residues are involved in the binding site.

1. Rat pancreatic islets were isolated and then maintained in culture for 2-4 days before being incubated in groups of 100 in the presence of different glucose (0-20 mM) or CaCl2 (1.2-4.2 mM) concentrations, or with uncoupler. 2. Increases in extracellular glucose concentration resulted in increases in the amount of active, non-phosphorylated, pyruvate dehydrogenase in the islets, with half-maximal effects around 5-6 mM-glucose. Increasing extracellular glucose from 3 to 20 mM resulted in a 4-6-fold activation of pyruvate dehydrogenase within 2 min. 3. The total enzyme activity was unchanged, and averaged 0.4 m-unit/100 islets at 37 degrees C. 4. These changes in active pyruvate dehydrogenase were broadly similar to changes in insulin secretion by the islets. 5. Increasing extracellular Ca2+ or adding uncoupler also activated pyruvate dehydrogenase to a similar degree, but only the former was associated with increased insulin secretion.     

Studies on the biotin-binding sites of avidin and streptavidin. A chemically induced dynamic nuclear polarization investigation of the status of tyrosine residues.

We applied the protein photochemically induced dynamic nuclear polarization (photo-c.i.d.n.p.) method to explore the conformation of the side chains of tyrosine, tryptophan and histidine residues in three biotin-binding proteins. The c.i.d.n.p. spectra of avidin, streptavidin and 'core' streptavidin were compared with those of their complexes with biotin and its derivatives. The data indicate that the single tyrosine residue (Tyr-33) of avidin is clearly inaccessible to the triplet flavin photo-c.i.d.n.p. probe. The same holds for all tryptophan and histidine side chains. Although the analogous Tyr-43 residue of streptavidin is also buried, at least three of the other tyrosine residues of this protein are exposed. The same conclusions apply to the truncated form of the protein, core streptavidin. As judged by the photo-c.i.d.n.p. results, complexing of avidin and streptavidin with biotin, N-epsilon-biotinyl-L-lysine (biocytin) or biotinyltyrosine has little or no effect on tyrosine accessibility in these proteins. Biotinyltyrosine can be used to probe the depth of the corresponding binding site. The accessibility of the tyrosine side chain of biotinyltyrosine in the complex demonstrates the exquisite fit of the biotin-binding cleft of avidin: only the biotin moiety appears to be accommodated, leaving the tyrosine side chain exposed.     

Studies on the catalytic residues at the active site of human liver alpha-L-fucosidase

Kinetic studies and chemical modifications were performed on purified human liver alpha-L-fucosidase (alpha-L-fucoside fucohydrolase, EC 3.2.1.51) in an attempt to identify the catalytic residues at the active site. Plots of log Vmax vs. pH (computer-fitted to a theoretical model) displayed two apparent pK values, of approx. 3.8 and 7.3. The temperature dependence of these pK values yielded heats of ionization of 3 and 0 kcal/mol from Van't Hoff plots for the lower and higher pK values, respectively. Reaction of alpha-L-fucosidase with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and sodium p-(hydroxymercuri)benzoate resulted in complete inactivation of the enzyme. Other nonspecific inactivators had little or no effect on enzyme activity. These results suggest two carboxyl groups whose ionization state is important to activity, a non-active-site cysteine residue important to activity, and at least one active-site carboxyl group.     

Environmental effects on the protonation states of active site residues in bacteriorhodopsin.

Finite difference solutions of the Poisson-Boltzmann equation are used to calculate the pKa values of the functionally important ionizable groups in bacteriorhodopsin. There are strong charge-charge interactions between the residues in the binding site leading to the possibility of complex titration behavior. Structured water molecules, if they exist in the binding site, can have significant effects on the calculated pKa by strongly stabilizing ionized species. The ionization states of the Schiff base and Asp-85 are found to be strongly coupled. Small environmental changes, which might occur as a consequence of trans-cis isomerization, are capable of causing large shifts in the relative pKa values of these two groups. This provides an explanation for the protonation of Asp-85 and the deprotonation of the Schiff base in the M state of bacteriorhodopsin. The different behavior of Asp-85 and Asp-212 is discussed in this regard.     

Effects of mutations of the active site arginine residues in 4-oxalocrotonate tautomerase on the pKa values of active site residues and on the pH dependence of catalysis.

The unusually low pK(a) value of the general base catalyst Pro-1 (pK(a) = 6.4) in 4-oxalocrotonate tautomerase (4-OT) has been ascribed to both a low dielectric constant at the active site and the proximity of the cationic residues Arg-11 and Arg-39 [Stivers, J. T., Abeygunawardana, C., Mildvan, A. S., Hajipour, G., and Whitman, C. P. (1996) Biochemistry 35, 814-823]. In addition, the pH-rate profiles in that study showed an unidentified protonated group essential for catalysis with a pK(a) of 9.0. To address these issues, the pK(a) values of the active site Pro-1 and lower limit pK(a) values of arginine residues were determined by direct (15)N NMR pH titrations. The pK(a) values of Pro-1 and of the essential acid group were determined independently from pH-rate profiles of the kinetic parameters of 4-OT in arginine mutants of 4-OT and compared with those of wild type. The chemical shifts of all of the Arg Nepsilon resonances in wild-type 4-OT and in the R11A and R39Q mutants were found to be independent of pH over the range 4.9-9.7, indicating that no arginine is responsible for the kinetically determined pK(a) of 9.0 for an acidic group in free 4-OT. With the R11A mutant, where k(cat)/K(m) was reduced by a factor of 10(2.9), the pK(a) of Pro-1 was not significantly altered from that of the wild-type enzyme (pK(a) = 6.4 +/- 0.2) as revealed by both direct (15)N NMR titration (pK(a) = 6.3 +/- 0.1) and the pH dependence of k(cat)/K(m) (pK(a) = 6.4 +/- 0.2). The pH-rate profiles of both k(cat)/K(m) and k(cat) for the reaction of the R11A mutant with the dicarboxylate substrate, 2-hydroxymuconate, showed humps, i.e., sharply defined maxima followed by nonzero plateaus. The humps disappeared in the reaction with the monocarboxylate substrate, 2-hydroxy-2,4-pentadienoate, indicating that, unlike the wild-type enzyme which reacts only with the dianionic form of the dicarboxylic substrate, the R11A mutant reacts with both the 6-COOH and 6-COO(-) forms, with the 6-COOH form being 12-fold more active. This reversal in the preferred ionization state of the 6-carboxyl group of the substrate that occurs upon mutation of Arg-11 to Ala provides strong evidence that Arg-11 interacts with the 6-carboxylate of the substrate. In the R39Q mutant, where k(cat)/K(m) was reduced by a factor of 10(3), the kinetically determined pK(a) value for Pro-1 was 4.6 +/- 0.2, while the ionization of Pro-1 showed negative cooperativity with an apparent pK(a) of 7.1 +/- 0.1 determined by 1D (15)N NMR. From the Hill coefficient of 0.54, it can be shown that the apparent pK(a) value of 7.1 could result most simply from the averaging of two limiting pK(a) values of 4.6 and 8.2. Mutation of Arg-39, by altering the structure of the beta-hairpin which covers the active site, could result in an increase in the solvent exposure of Pro-1, raising its upper limit pK(a) value to 8.2. In the R39A mutant, the kinetically determined pK(a) of Pro-1 was also low, 5.0 +/- 0.2, indicating that in both the R39Q and R39A mutants, only the sites with low pK(a) values were kinetically operative. With the fully active R61A mutant, the kinetically determined pK(a) of Pro-1 (pK(a) = 6.5 +/- 0.2) agreed with that of wild-type 4-OT. It is concluded that the unusually low pK(a) of Pro-1 shows little contribution from electrostatic effects of the nearby cationic Arg-11, Arg-39, and Arg-61 residues but results primarily from a site of low local dielectric constant.     

Effect of active site residues in barnase on activity and stability.

We have mutated residues in the active site of the ribonuclease, barnase, in order to determine their effects on both enzyme activity and protein stability. Mutation of several of the positively charged residues that interact with the negatively charged RNA substrate (Lys27----Ala, Arg59----Ala and His102----Ala) causes large decreases in activity. This is accompanied, however, by an increase in stability. There is presumably electrostatic strain in the active site where positively charged side-chains are clustered. Mutation of several residues that make hydrogen bonds (Ser57----Ala, Asn58----Asp and Tyr103----Phe) causes smaller decreases in activity, but increases or has no effect on stability. Deletion of hydrogen bonding groups elsewhere in proteins has been found previously to decrease stability by 0.5 to 1.5 kcal mol-1. Conversely, we find that two mutations (Asp54----Asn and Gln104----Ala) decrease stability and increase activity. Another mutation (Glu73----Ala) decreases both activity and stability. It is clear that many residues in the active site do not contribute to stability and that for some, but not all, of the residues there is a compromise between activity and stability. This suggests that certain types of local instability may be necessary for substrate binding and catalysis by barnase. This has implications for the understanding of enzyme activity and the design of enzymes.     

Functional residues at the active site of angiotensin converting enzyme.

A series of chemical modification reactions have been carried out with rabbit pulmonary angiotensin converting enzyme (dipeptidyl carboxypeptidase, EC 3.4.15.1) in order to identify amino acid residues essential for its catalytic activity. The enzyme is rapidly inactivated by nitration with tetranitromethane and by O-acetylation with N-acetylimidazole. Deacylation with hydroxylamine restores activity to the acetylated enzyme, while the inhibitor, β-phenylpropionyl-L-phenylalanine, protects against acetylimidazole inactivation. These results indicate the presence of functional tyrosyl residues at the active site of the enzyme. Reaction with butanedione decreases activity, an effect that is markedly enhanced by the presence of borate, indicating essential arginyl residues. In addition, activity is diminished by the carboxyl reagent, cyclohexylmorpholinoethyl carbodiimide. Thus, the three functional residues long known to be components of the active site of bovine carboxypeptidase A, tyrosyl, arginyl, and glutamyl, have counterparts in the angiotensin converting enzyme. The effects of pyridoxal phosphate and a number of other reagents demonstrate that the converting enzyme also contains an important lysyl residue.     

Site-directed mutagenesis of active site residues in Bacillus subtilis alpha-amylase.

Site-directed mutagenesis of Bacillus subtilis N7 alpha-amylase has been performed to evaluate the roles of the active site residues in catalysis and to prepare an inactive catalytic-site mutant that can form a stable complex with natural substrates. Mutation of Asp-176, Glu-208, and Asp-269 to their amide forms resulted in over a 15,000-fold reduction of its specific activity, but all the mutants retained considerable substrate-binding abilities as estimated by gel electrophoresis in the presence of soluble starch. Conversion of His-180 to Asn resulted in a 20-fold reduction of kcat with a 5-fold increase in Km for a maltopentaose derivative. The relative affinities for acarbose vs. maltopentaose were also compared between the mutants and wild-type enzyme. The results are consistent with the roles previously proposed in Taka-amylase A and porcine pancreatic alpha-amylase based on their X-ray crystallographic analyses, although different pairs had been assigned as catalytic residues for each enzyme. Analysis of the residual activity of the catalytic-site mutants by gel electrophoresis has suggested that it derived from the wild-type enzyme contaminating the mutant preparations, which could be removed by use of an acarbose affinity column; thus, these mutants are completely devoid of activity. The affinity-purified mutant proteins should be useful for elucidating the complete picture of the interaction of this enzyme with starch.     

Identification of active site residues in Bradyrhizobium japonicum acetyl-CoA synthetase.

Acetyl-CoA synthetase (ACS) catalyses the activation of acetate to acetyl-CoA in the presence of ATP and CoA. The gene encoding Bradyrhyzobium japonicum ACS has been cloned, sequenced, and expressed in Escherichia coli. The enzyme comprises 648 amino acid residues with a calculated molecular mass of 71,996 Da. The recombinant enzyme was also purified from the transformed E. coli. The enzyme was essentially indistinguishable from the ACS of B. japonicum bacteroids as to the criteria of polyacrylamide gel electrophoresis and biochemical properties. Based on the results of database analysis, Gly-263, Gly-266, Lys-269, and Glu-414 were selected for site-directed mutagenesis in order to identify amino acid residues essential for substrate binding and/or catalysis. Four different mutant enzymes (G263I, G266I, K269G, and E414Q) were prepared and then subjected to steady-state kinetic studies. The kinetic data obtained for the mutants suggest that Gly-266 and Lys-269 participate in the formation of acetyl-AMP, whereas Glu-414 may play a role in acetate binding.     

Studies on determination of active site amino acid residues in glyoxylate synthetase from potato tuber chloroplasts

A homogeneous preparation of glyoxylate synthetase from greening potato tubers was used to study the functional role of disulphide groups, lysine and tryptophan residues in enzyme catalysis. The formation of a thioisoindole derivative was demonstrated by spectral analysis of the reduced and o-phthalaldehyde-treated enzymes. o-Phthalaldehyde modification resulted in about a 25 % loss of tryptophan emission at 336 nm and the appearance of a 410-nm emission peak characteristic of a thioisoindole. Ferrous iron was capable of generating thiol groups and addition of substrate resulted in a faster disappearance of these thiols. The optimal time for maximum glyoxylate synthesis by glyoxylate synthetase paralleled the disappearance of these thiols. Involvement of lysine and tryptophan residues in the enzyme reaction was demonstrated by the inhibition of activity by pyridoxal 5′-phosphate and dimethyl(2-hydroxy 5-nitrobenzyl) sulphonium bromide (DMHNB), respectively. Pyridoxal phosphate strongly and reversibly inhibited glyoxylate synthetase, and substrate and metal ion provided significant protection against inhibition. The results suggest that the lysine residue may be at or near the active binding site. The lysyl residue formed a Schiff base with pyridoxal phosphate which was stabilised by NaBH₄. Glyoxylate synthetase was also irreversibly inactivated by a tryptophan selective reagent, DMHNB, while substrate provided substantial protection against inactivation. Kinetic analysis and correlation of the spectral data at 410 nm indicated that complete inactivation by DMHNB resulted from the modification of 5 tryptophan residues/subunit, of which one was essential for activity. The available evidence suggests a possible concerted action of enzyme disulphides, ferrous iron, lysine and aromatic amino acid residues in the synthesis of glyoxylate by this enzyme.     

Mutational analysis of active site residues in pig heart aconitase.

A cDNA encoding mature porcine heart aconitase was over-expressed in Escherichia coli under the control of a phage T7 promoter. Recombinant aconitase purified from E. coli was identical to the enzyme from pig and beef heart in size, [3Fe-4S] and [4Fe-4S] cluster structure and enzymatic activity. Nine amino acid residues in close proximity to the Fe-S cluster and bound substrate (Lauble, H., Kennedy, M.C., Beinert, H., and Stout, C.D. (1992) Biochemistry, in press) were replaced by site-directed mutagenesis. Fe-S cluster environment as indicated by the EPR spectrum, tight binding of substrate, and enzymatic activity were compared for the mutant and wild type enzymes. Significant perturbations were detected for all of the mutant enzymes. Replacements for Asp100, His101, Asp165, Arg580, and Ser642 result in a 10(3)-10(5)-fold drop in activity, which suggests that these residues are involved in critical aspects of the reaction. Arg580 appears to be a key residue for substrate binding, as shown by a 30-fold increased Km and loss of tight substrate binding. Results of mutagenesis support the interpretation of the x-ray model, namely that Asp100 and His101 form an ion pair for elimination of the substrate hydroxyl and Ser642 may function as a general base for proton abstraction from citrate or isocitrate in the dehydration step and protonation of cis-aconitate in the hydration step. Asp165 appears to play a critical role in the interaction of Fea with substrate.