Active site histidine

Fig. 10.12 Sequence alignment of trypsin, chymotrypsin and thrombin (bovine). The active sites histidine, aspartic acid and serine are highlighted.

The mechanism of succinyl-CoA synthetase is postulated to involve displacement of CoA by phosphate, forming succinyl phosphate at the active site, followed by transfer of the phosphoryl group to an active-site histidine (making a phosphohistidine intermediate) and release of succinate. The phosphoryl moiety is then transferred to GDP to form GTP (Figure 20.13). This sequence of steps preserves the energy of the thioester bond of succinyl-CoA in a series of high-energy intermediates that lead to a molecule of ATP ... 

Pelton JG, Torchia DA, Meadow ND, Roseman S. Tautomeric states of the active-site histidines of phosphorylated and unphosphorylated IIIGlc, a signal-transuding protein from Escherichia coli, using two-dimensional heteronuclearNMR techniques. Prot Sci 1993 2 543-558. 

This mechanism has been supported by x-ray studies on crystallized xylose isomerase that suggest that the "active-site histidine" (presumed to be His 53, or 54 in some sequences) is located correctly so as to properly remove a proton from either Cl or C2 of the substrate, and that two threonine residues (Thr90 and Thr91, or 89,90 in some sequences) are positioned to provide polar oxygens to be either the acceptor or the donor proton in the cu-enediol configuration (57). 

In isoenzyme I, the titration behavior of zinc H20 is complicated due to the presence of three titratable active-site histidines as described in Section VI.D. Lindskog reports a value of 7.1 for the pKa of zinc-H20 (142), but higher values were obtained by other authors. The pH-rate profile for 4-nitrophenyl acetate esterase activity yields pKa = 7.45 (142). 

Similar isotope effects for human isoenzyme I (157c) on kc t and Km for C02 hydration are 1.7. Silverman and Tu (161) report an isotope effect of 2.5 for H2180 release and suggest that the intrinsic isotope effect of intramolecular H+ transfer might be significantly smaller in isoenzyme I than in isoenzyme II. Hence, the H20-splitting step might also limit the rate of C02 hydration in isoenzyme I. Human isoenzyme I has three titratable active-site histidines with pKa values... 

Ginsburg, A. Szczepanowski, R.H. Ruvinov, S.B. Nosworthy, N.J. Sondej, M. Umland, T.C. Peterkofsky, A. Conformational stability changes of the amino terminal domain of enzyme I of the Escherichia coli phosphoenolpyruvate sugar phosphotransferase system produced by substituting alanine or glutamate for the active-site histidine 189 implications for phosphorylation effects. Protein Sci., 9, 1085-1094 (2000)... 

Napper, S. Delbaere, L.T.J. Waygood, E.B. The aspartyl replacement of the active site histidine in histidine-containing protein, HPr, of the Escherichia coli phosphoenolpyruvate sugar phosphotransferase system can accept and donate a phosphoryl group. Spontaneous dephosphorylation of acyl-phosphate autocatalyzes an internal cyclization. J. Biol. Chem., 274, 21776-21782 (1999)... 

Support for this concept is provided by H NMR studies which have identified a downfield resonance of the hydrogen-bonded proton in this pair at 18 ppm in chymotrypsinogen and chymotrypsin at low pH and at 14.9-15.5 ppm at high pH values.246 247 Similar resonances are seen in the a-lytic protease,248 in sub-tilisin,249 in adducts of serine proteases with boronic acids250 251 or peptidyl trifluoromethyl ketones,252 in alkylated derivative of the active site histidine,253 and in molecular complexes that mimic the Asp-His pair in the active sites of serine proteases.254... 

Most kinases transfer chiral phospho groups with inversion and fail to catalyze partial exchange reactions that would indicate phosphoenzyme intermediates. However, nucleoside diphosphate kinase contains an active site histidine which is phosphorylated to form a phosphoenzyme.869 The enzyme catalyzes phosphorylation of nucleoside diphosphates other than ADP by a nucleotide triphosphate, usually ATP. 

N NMR has also been used to measure the pKa value of the active-site histidine of the serine proteases,19 confirming that it is the histidine and not the buried aspartate that ionizes with a pKa of about 7. 

Montero-Moran GM, Lara-Gonzalez S, Alvarez-Anorve LI et al (2001) On the multiple functional roles of the active site histidine in catalysis and allosteric regulation of Escherichia coli glucosamine 6-phosphate deaminase. Biochemistry 40 10187-10196... 

Geurtsen, J., Steeghs, L., Hove, J.T., van der Ley, P., Tommassen, J. Dissemination of lipid A dea-cylases (pagL) among gram-negative bacteria identification of active-site histidine and serine residues. J Biol Chem 280 (2005) 8248-8259. 

Figure 12.18 shows a portion of the Fi = Hn, F = region of the NOESY spectrum of B. subtilis HPr, a phosphotransferase that uses an active-site histidine side chain to transfer a high-energy phosphate group. A large number of sequential U (i) Hn (i + 1) NOE crosspeaks are shown. The Hn chemical shifts are indicated by vertical dotted... 

Peptide Chloromethyl Ketones. Peptide chloromethyl ketone inhibitors have been studied extensively and a fairly detailed picture of the inhibition reaction (see Figure 3) has emerged from numerous chemical and crystallographic studies (30,31). The inhibitor resembles a serine protease substrate with the exception that the scissile peptide bond of the substrate is replaced with a chloromethyl ketone functional group in the inhibitor. The inhibitor binds to the serine protease in the extended substrate binding site and the reactive chloromethyl ketone functional group is placed then in the proper position to alkylate the active-site histidine residue. In addition, the serine OH reacts with the inhibitor carbonyl group to form a hemiketal. 

R, Olsen, J.G., McGuire, K.A., and Henriksen, A., Fatty acid synthesis. Role of active site histidines and lysine in Cys-His-His-type beta-ketoacyl-acyl carrier protein synthases, FEBS J. 273, 695-710, 2(X)6 Ryn, Y, Kim, KJ., Rosennser, C.A., and Scott, A., Decarboxylative Qaisen condensation catalyzed by in vitro selected ribozymes, Chem. Commun. 7, 1439-1441, 2006. 

Reaction at active site histidine residues of trypsinlike serine proteases. 

---