Kinetic analysis of the zinc-dependent deacetylase in the lipid A biosynthetic pathway

The first committed step of lipid A biosynthesis in Gram-negative bacteria is catalyzed by the zinc-dependent hydrolase LpxC that removes an acetate from the nitrogen at the 2' '-position of UDP-3-O-acyl-N-acetylglucosamine. Recent structural characterization by both NMR and X-ray crystallography provides many important details about the active site environment of LpxC from Aquifex aeolicus, a heat-stable orthologue that displays 32% sequence identity to LpxC from Escherichia coli. The detailed reaction mechanism and specific roles of active site residues for LpxC from A. aeolicus are further analyzed here. The pH dependencies of k(cat)/K(M) and k(cat) for the deacetylation of the substrate UDP-3-O-[(R)-3-hydroxymyristoyl]-GlcNAc are both bell-shaped. The ascending acidic limb (pK(1)) was fitted to 6.1 +/- 0.2 for k(cat) and 5.7 +/- 0.2 for k(cat)/K(M). The descending basic limb (pK(2)) was fitted to 8.0 +/- 0.2 for k(cat) and 8.4 +/- 0.2 for k(cat)/K(M). The pH dependence of the E73A mutant exhibits loss of the acidic limb, and the mutant retains only 0.15% activity versus the wild type. The pH dependencies of the other active site mutants H253A, K227A, H253A/K227A, and D234N remain bell-shaped, although their significantly lower activities (0.25%, 0.05%, 0.007%, and 0.57%, respectively) suggest that they contribute significantly to catalysis. Our cumulative data support a mechanism for LpxC wherein Glu73 serves as the general base for deprotonation and activation of the zinc-bound water.     

Insensitivity of perturbed carboxyl pK(a) values in the ovomucoid third domain to charge replacement at a neighboring residue

A number of carboxyl groups in turkey ovomucoid third domain (OMTKY3) have low pK(a) values. A previous study suggested that neighboring amino groups were primarily responsible for the low carboxyl pK(a) values. However, the expected elevation in pK(a) values for these amino groups was not observed. In the present study, site-directed mutagenesis is used to investigate the origins of perturbed carboxyl pK(a) values in OMTKY3. Electrostatic calculations suggest that Lys 34 has large effects, 0.4-0.6 unit, on Asp 7, Glu 10, and Glu 19 which are 5-11 A away from Lys 34. Two-dimensional (1)H NMR techniques were used to determine pK(a) values of the acidic residues in OMTKY3 mutants in which Lys 34 has been replaced with threonine and glutamine. Surprisingly, the pK(a) values in the mutants are very close to those of the wild-type protein. The insensitivity of the acidic residues to replacement of Lys 34 suggests that long-range electrostatic interactions play less of a role in perturbing carboxyl pK(a) values than originally thought. We hypothesize that hydrogen bonds play a key role in perturbing some of the carboxyl ionization equilibria in OMTKY3.     

NMR determination of pKa values in α-synuclein

The intrinsically unfolded protein α-synuclein has an N-terminal domain with seven imperfect KTKEGV sequence repeats and a C-terminal domain with a large proportion of acidic residues. We characterized pK(a) values for all 26 sites in the protein that ionize below pH 7 using 2D (1) H-(15) N HSQC and 3D C(CO)NH NMR experiments. The N-terminal domain shows systematically lowered pK(a) values, suggesting weak electrostatic interactions between acidic and basic residues in the KTKEGV repeats. By contrast, the C-terminal domain shows elevated pK(a) values due to electrostatic repulsion between like charges. The effects are smaller but persist at physiological salt concentrations. For α-synuclein in the membrane-like environment of sodium dodecylsulfate (SDS) micelles, we characterized the pK(a) of His50, a residue of particular interest since it is flanked within one turn of the α-helix structure by the Parkinson's disease-linked mutants E46K and A53T. The pK(a) of His50 is raised by 1.4 pH units in the micelle-bound state. Titrations of His50 in the micelle-bound states of the E46K and A53T mutants show that the pK(a) shift is primarily due to interactions between the histidine and the sulfate groups of SDS, with electrostatic interactions between His50 and Glu46 playing a much smaller role. Our results indicate that the pK(a) values of uncomplexed α-synuclein differ significantly from random coil model peptides even though the protein is intrinsically unfolded. Due to the long-range nature of electrostatic interactions, charged residues in the α-synuclein sequence may help nucleate the folding of the protein into an α-helical structure and confer protection from misfolding.     

Glutamic acid in the inhibitory site of mitochondrial ATPase inhibitor, IF(1), participates in pH sensing in both mammals and yeast

The mitochondrial ATPase inhibitor, IF(1), regulates the activity of F(1)F(o)-ATPase. The inhibitory activity of IF(1) is highly pH-dependent. The effective inhibition by IF(1) requires a low pH. Under basic conditions, its activity markedly declines. The importance of His49 in the pH dependence of bovine IF(1) is well-known. However, the residue is not conserved in yeast IF(1). We previously showed that Glu21 is required for the pH dependence of yeast IF(1), but the function of homologous Glu in mammalian IF(1) is not clear. In this study, we examined the requirement for Glu26 of bovine IF(1) (corresponding to Glu21 of yeast IF(1)) regarding its pH dependence by amino acid replacement. Three mutant proteins, E26A, H49K and the double mutant E26A/H49K, were overexpressed and purified. All mutants retained their inhibitory activity well at pH 8.2, although wild-type IF(1) was approximately 10-fold less active at pH 8.2 than at 6.5. A covalent cross-linking study revealed that both wild-type IF(1) and the E26A mutant formed a tetramer at pH 8.2, although H49K and E26A/H49K mutants did not. These results indicate that, in addition to His49, Glu26 participates in pH sensing in bovine IF(1), and the mechanism of pH sensing mediated by Glu26 is different from the dimer-tetramer model proposed previously.     

pH Dependence of the reaction catalyzed by avian mitochondrial phosphoenolpyruvate carboxykinase

The pH dependence of the reaction catalyzed by phosphoenolpyruvate carboxykinase (PEPCK) provides significant insight into the chemical mechanism. The pH dependence of k(cat) shows the importance of two acidic ionizations with pK(a) values of 6.5 and 7.0 assigned to the active site metal ligands H249 and K228. A single basic ionization is observed with an apparent pK(a) value of 8.4 that is assigned to K275 that is located in the P-loop motif and is essential for phosphoryl transfer. The pH dependence of k(cat)/K(M,PEP) demonstrates the importance of the same two acidic ionizations in the interaction of phosphoenolpyruvate with PEPCK and a single basic ionization with a pK(a) value of 8.1 that is assigned to Y220. The interaction of Mg-IDP with PEPCK is dependent upon a single acidic ionization attributed to K228 and two basic ionizations, both having an average pK(a) value of 8.1. One of the basic ionizations is attributed to the P-loop lysine (K275) and the other to C273.     

Mimicking of K+ activation by double mutation of glutamate 795 and glutamate 820 of gastric H+,K+-ATPase

Six double mutants of Glu(795) and Glu(820) present in transmembrane domains 5 and 6 of the alpha-subunit of rat gastric H(+),K(+)-ATPase were generated and expressed with the baculovirus expression system. Five of the six mutants exhibited an SCH 28080-sensitive ATPase activity in the absence of K(+). The activity levels decreased in the following order: E795Q/E820A > E795Q/E820Q > E795Q/E820D congruent with E795A/E820A > E795L/E820Q. The E795L/E820D mutant possessed no constitutive activity. The relative low ATPase activity of the E795L/E820Q mutant is due to its low phosphorylation rate so that the dephosphorylation step was no longer rate-limiting. The constitutively active mutants showed a much lower vanadate sensitivity than the wild-type enzyme and K(+)-sensitive mutants, indicating that these mutants have a preference for the E(1) conformation. In contrast to the constitutively active single mutants generated previously, the double mutants exhibited a high spontaneous dephosphorylation rate at 0 degrees C compared to that of the wild-type enzyme. In addition, the H(+),K(+)-ATPase inhibitor SCH 28080 increased the steady-state phosphorylation level of the constitutively active mutants, due to the formation of a stable complex with the E(2)-P form. These studies further substantiate the idea that the empty ion binding pockets of some mutants apparently mimic the K(+)-filled binding pocket of the native enzyme.     

The carbonyl group of glutamic acid-795 is essential for gastric H+,K+-ATPase activity

To study the role of Glu795offresent in the fifth transmembrane domain of the alpha-subunit of gastric H+,K+-ATPase, several mutants were generated and expressed in Sf9 insect cells. The E795Q mutant had rather similar properties as the wild-type enzyme. The apparent affinity for K+ in both the ATPase reaction and the dephosphorylation of the phosphorylated intermediate was even slightly enhanced. This indicates that the carbonyl group of Glu795 is sufficient for enzymatic activity. This carbonyl group, however, has to be at a particular position with respect to the other liganding groups, since the E795D and E795N mutants showed a strongly reduced ATPase activity, a lowered apparent K+ affinity, and a decreased steady-state phosphorylation level. In the absence of a carbonyl residue at position 795, the K+ sensitivity was either strongly decreased (E795A) or completely absent (E795L). The mutant E795L, however, showed a SCH 28080 sensitive ATPase activity in the absence of K+, as well as an enhanced spontaneous dephosphorylation rate, that could not be further enhanced by K+, suggesting that this mutant mimicks the filled K+ binding pocket. The results indicate that the Glu795 residue is involved in K+-stimulated ATPase activity and K+-induced dephosphorylation of the phosphorylated intermediate. Glu795 might also be involved in H+ binding during the phosphorylation step, since the mutants E795N, E795D, and E795A showed a decrease in the phosphorylation rate as well as in the apparent ATP affinity in the phosphorylation reaction. This indicates that Glu795 is not only involved in K+ but might also play a role in H+ binding.     

Mechanism of the family 1 beta-glucosidase from Streptomyces sp: catalytic residues and kinetic studies

The Streptomyces sp. beta-glucosidase (Bgl3) is a retaining glycosidase that belongs to family 1 glycosyl hydrolases. Steady-state kinetics with p-nitrophenyl beta-D-glycosides revealed that the highest k(cat)/K(M) values are obtained with glucoside (with strong substrate inhibition) and fucoside (with no substrate inhibition) substrates and that Bgl3 has 10-fold glucosidase over galactosidase activity. Reactivity studies by means of a Hammett analysis using a series of substituted aryl beta-glucosides gave a biphasic plot log k(cat) vs pK(a) of the phenol aglycon: a linear region with a slope of beta(lg) = -0.8 for the less reactive substrates (pK(a) > 8) and no significant dependence for activated substrates (pK(a) < 8). Thus, according to the two-step mechanism of retaining glycosidases, formation of the glycosyl-enzyme intermediate is rate limiting for the former substrates, while hydrolysis of the intermediate is for the latter. To identify key catalytic residues and on the basis of sequence similarity to other family 1 beta-glucosidases, glutamic acids 178 and 383 were changed to glutamine and alanine by site-directed mutagenesis. Mutation of Glu178 to Gln and Ala yielded enzymes with 250- and 3500-fold reduction in their catalytic efficiencies, whereas larger reduction (10(5)-10(6)-fold) were obtained for mutants at Glu383. The functional role of both residues was probed by a chemical rescue methodology based on activation of the inactive Ala mutants by azide as exogenous nucleophile. The E178A mutant yielded the beta-glucosyl azide adduct (by (1)H NMR) with a 200-fold increase on k(cat) for the 2,4-dinitrophenyl glucoside but constant k(cat)/K(M) on azide concentration. On the other hand, the E383A mutant with the same substrate gave the alpha-glucosyl azide product and a 100-fold increase in k(cat) at 1 M azide. In conclusion, Glu178 is the general acid/base catalyst and Glu383 the catalytic nucleophile. The results presented here indicate that Bgl3 beta-glucosidase displays kinetic and mechanistic properties similar to other family 1 enzymes analyzed so far. Subtle differences in behavior would lie in the fine and specific architecture of their respective active sites.     

Functional importance of charged residues within the putative intracellular loops in pH regulation by Na+/ H+ exchanger NHE1

The plasma membrane Na+/H+ exchanger 1 is activated in response to various extrinsic factors, and this process is regulated by an intracellular pH-sensing mechanism. To identify the candidate residues responsible for intracellular pH regulation, we analyzed the functional properties of engineered Na+/H+ exchanger 1 mutants with charge-reversal mutations of charged residues located in the intracellular loops. Na+/H+ exchanger 1 mutants with mutations at 11 positions were well expressed in the plasma membrane, but that with E247R was not, suggesting that Glu247 is important for the functional expression of Na+/H+ exchanger 1. Charge-reversal mutations of Glu131 (E131R, E131K) and Arg327 (R327E) resulted in a shift in the intracellular pH dependence of the exchange activity measured by 22Na+ uptake to the acidic side, and it abolished the response to growth factors and a hyperosmotic medium; however, mutations of Asp448 (D448R) and Arg500 (R500E) slightly shifted it to the alkaline side. In E131R, in addition to the change in intracellular pH dependence, the affinities for extracellular Na+, Li+ and the inhibitor 5-(N-ethyl-N-isopropyl)amiloride significantly increased. Furthermore, charge-conserved mutation of E131 (E131D) was found to have no effect, whereas charge neutralization (E131Q) resulted in a slight acidic shift of exchange. These results support the view that the multiple charged residues identified in this study, along with several basic residues reported previously, participate in the regulation of the intracellular pH sensing of Na+/H+ exchanger 1. In addition, Glu131 may also be important for cation transport.     

A rationalization of the acidic pH dependence for stromelysin-1 (Matrix metalloproteinase-3) catalysis and inhibition

The pH dependence of matrix metalloproteinase (MMP) catalysis is described by a broad bell-shaped curve, indicating the involvement of two unspecified ionizable groups in proteolysis. Stromelysin-1 has a third pK(a) near 6, resulting in a uniquely sharp acidic catalytic optimum, which has recently been attributed to His(224). This suggests the presence of a critical, but unidentified, S1' substructure. Integrating biochemical characterizations of inhibitor-enzyme interactions with active site topography from corresponding crystal structures, we isolated contributions to the pH dependence of catalysis and inhibition of active site residues Glu(202) and His(224). The acidic pK(a) 5.6 is attributed to the Glu(202).zinc.H(2)O complex, consistent with a role for the invariant active site Glu as a general base in MMP catalysis. The His(224)-dependent substructure is identified as a tripeptide (Pro(221)-Leu(222)-Tyr(223)) that forms the substrate cleft lower wall. Substrate binding induces a beta-conformation in this sequence, which extends and anchors the larger beta-sheet of the enzyme. substrate complex and appears to be essential for productive substrate binding. Because the PXY tripeptide is strictly conserved among MMPs, this "beta-anchor" may represent a common motif required for macromolecular substrate hydrolysis. The striking acidic profile of stromelysin-1 defined by the combined ionization of Glu(202) and His(224) allows the design of highly selective inhibitors.     

The effect of net charge on the solubility, activity, and stability of ribonuclease Sa

The net charge and isoelectric pH (pI) of a protein depend on the content of ionizable groups and their pK values. Ribonuclease Sa (RNase Sa) is an acidic protein with a pI = 3.5 that contains no Lys residues. By replacing Asp and Glu residues on the surface of RNase Sa with Lys residues, we have created a 3K variant (D1K, D17K, E41K) with a pI = 6.4 and a 5K variant (3K + D25K, E74K) with a pI = 10.2. We show that pI values estimated using pK values based on model compound data can be in error by >1 pH unit, and suggest how the estimation can be improved. For RNase Sa and the 3K and 5K variants, the solubility, activity, and stability have been measured as a function of pH. We find that the pH of minimum solubility varies with the pI of the protein, but that the pH of maximum activity and the pH of maximum stability do not.     

Specific effects of chloride on the photocycle of E194Q and E204Q mutants of bacteriorhodopsin as measured by FTIR spectroscopy

Low-temperature Fourier transform infrared spectroscopy has been used to study mutants of Glu194 and Glu204, two amino acids that are involved in proton release to the extracellular side of bacteriorhodopsin. Difference spectra of films of E194Q, E204Q, E194Q/E204Q, E9Q/E194Q/E204Q, and E9Q/E74Q/E194Q/E204Q at 243, 277, and 293 K and several pH values were obtained by continuous illumination. A specific effect of Cl(-) ions was found for the mutants, promoting a N-like intermediate at alkaline pH and an O' intermediate at neutral or acid pH. The apparent pK(a) of Asp85 in the M intermediate was found to be decreased for E194Q in the presence of Cl(-) (pK(a) of 7.6), but it was unchanged for E204Q, as compared to wild-type. In the absence of Cl(-) (i.e., in the presence of SO(4)(2)(-)), mutation of Glu194 or of Glu204 produces M- (or M(N), M(G))-like intermediates under all of the conditions examined. The absence of N, O, and O' intermediates suggests a long-range effect of the mutation. Furthermore, it is suggested that Cl(-) acts by reaching the interior of the protein, rather than producing surface effects. The effect of low water content was also examined, in the presence of Cl(-). Similar spectra corresponding to the M(1) intermediate were found for dry samples of both mutants, indicating that the effects of the mutations or of Cl(-) ions are confined to the second part of the photocycle. The water O-H stretching data further confirms altered photocycles and the effect of Cl(-) on the accumulation of the N intermediate.     

Side-chain protonation and mobility in the sarcoplasmic reticulum Ca2+-ATPase: implications for proton countertransport and Ca2+ release

Protonation of acidic residues in the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA 1a) was studied by multiconformation continuum electrostatic calculations in the Ca(2+)-bound state Ca(2)E1, in the Ca(2+)-free state E2(TG) with bound thapsigargin, and in the E2P (ADP-insensitive phosphoenzyme) analog state with MgF(4)(2-) E2(TG+MgF(4)(2-)). Around physiological pH, all acidic Ca(2+) ligands (Glu(309), Glu(771), Asp(800), and Glu(908)) were unprotonated in Ca(2)E1; in E2(TG) and E2(TG+MgF(4)(2-)) Glu(771), Asp(800), and Glu(908) were protonated. Glu(771) and Glu(908) had calculated pK(a) values larger than 14 in E2(TG) and E2(TG+MgF(4)(2-)), whereas Asp(800) titrated with calculated pK(a) values near 7.5. Glu(309) had very different pK(a) values in the Ca(2+)-free states: 8.4 in E2(TG+MgF(4)(2-)) and 4.7 in E2(TG) because of a different local backbone conformation. This indicates that Glu(309) can switch between a high and a low pK(a) mode, depending on the local backbone conformation. Protonated Glu(309) occupied predominantly two main, very differently orientated side-chain conformations in E2(TG+MgF(4)(2-)): one oriented inward toward the other Ca(2+) ligands and one oriented outward toward a protein channel that seems to be in contact with the cytoplasm. Upon deprotonation, Glu(309) adopted completely the outwardly orientated side-chain conformation. The contact of Glu(309) with the cytoplasm in E2(TG+MgF(4)(2-)) makes this residue unlikely to bind lumenal protons. Instead it might serve as a proton shuttle between Ca(2+)-binding site I and the cytoplasm. Glu(771), Asp(800), and Glu(908) are proposed to take part in proton countertransport.     

Reverse protonation is the key to general acid-base catalysis in enolase

The pH dependence of enolase catalysis was studied to understand how enolase is able to utilize both general acid and general base catalysis in each direction of the reaction at near-neutral pHs. Wild-type enolase from yeast was assayed in the dehydration reaction (2-phospho-D-glycerate --> phosphoenolpyruvate + H(2)O) at different pHs. E211Q, a site-specific variant of enolase that catalyzes the exchange of the alpha-proton of 2-phospho-D-glycerate but not the complete dehydration, was assayed in a (1)H/(2)H exchange reaction at different pDs. Additionally, crystal structures of E211Q and E168Q were obtained at 2.0 and 1.8 A resolution, respectively. Analysis of the pH profile of k(cat)/K(Mg) for wild-type enolase yielded macroscopic pK(a) estimates of 7.4 +/- 0.3 and 9.0 +/- 0.3, while the results of the pD profile of the exchange reaction of E211Q led to a pK(a) estimate of 9.5 +/- 0.1. These values permit estimates of the four microscopic pK(a)s that describe the four relevant protonation states of the acid/base catalytic groups in the active site. The analysis indicates that the dehydration reaction is catalyzed by a small fraction of enzyme that is reverse-protonated (i.e., Lys345-NH(2), Glu211-COOH), whereas the hydration reaction is catalyzed by a larger fraction of the enzyme that is typically protonated (i.e., Lys345-NH(3)(+), Glu211-COO(-)). These two forms of the enzyme coexist in a constant, pH-independent ratio. The structures of E211Q and E168Q both show virtually identical folds and active-site architectures (as compared to wild-type enolase) and thus provide additional support to the conclusions reported herein. Other enzymes that require both general acid and general base catalysis likely require reverse protonation of catalytic groups in one direction of the reaction.     

Differential effects of two mutations at arginine-234 in the alpha subunit of human pyruvate dehydrogenase.

The most common mutation in the alpha subunit of the pyruvate dehydrogenase (E1) component of the human pyruvate dehydrogenase complex (PDC) is arginine-234 to glycine and glutamine in 12 and 3 patients, respectively. Interestingly, these two mutations at the same amino acid position cause E1 (and hence PDC) deficiency by apparently different mechanisms. Recombinant human R234Q E1 had similar V(max) (25.7 +/- 4.4 units/mg E1) and apparent K(m) (101 +/- 4 nM) values for TPP as recombinant wild-type human E1, while R234G E1 had no significant change in V(max) (33.6 +/- 4.7 units/mg E1) but had a 7-fold increase in its apparent K(m) value for TPP (497 +/- 25 nM). Both of the R234 mutant proteins had similar apparent K(m) values for pyruvate. Both R234Q and R234G mutant proteins displayed similar phosphorylation rates of sites 1 and 2 by pyruvate dehydrogenase kinase 2 (PDK2) and site 3 by PDK1 compared to wild-type E1. Phosphorylated R234Q E1, R234G E1, and wild-type E1 also had similar dephosphorylation rates of sites 1 and 2 by phosphopyruvate dehydrogenase phosphatase 1. The rate of dephosphorylation of site 3 was about 50% for R234Q E1 and without a significant change for R234G E1 compared to the wild type. The data indicate that the patients with the R234G E1 mutation are symptomatic due to a decreased ability of this mutant protein to bind TPP, whereas the patients with the R234Q E1 mutation are symptomatic due to a decreased rate of dephosphorylation of site 3, hence keeping the enzyme in a phosphorylated/inactivated form.     

Carboxyl-terminal hydrophilic tail of a NhaP type Na+/H+ antiporter from cyanobacteria is involved in the apparent affinity for Na+ and pH sensitivity

Little information is available on the C-terminal hydrophilic tails of prokaryotic Na(+)/H(+) antiporters. To address functional properties of the C-terminal tail, truncation mutants in this domain were constructed. Truncation of C-terminal amino acid residues of NhaP1 type antiporter from Synechocystis PCC6803 (SynNhaP1) did not change the V(max) values, but increased the K(m) values for Na(+) and Li(+) about 3 to 15-fold. Truncation of C-terminal tail of a halotolerant cyanobacterium Aphanothece halophytica (ApNhaP1) significantly decreased the V(max) although it did not alter the K(m) values for Na(+). The C-terminal part of SynNhaP1 was expressed in E. coli and purified as a 16kDa soluble protein. Addition of purified polypeptide to the membrane vesicles expressing the C-terminal truncated SynNhaP1 increased the exchange activities. Change of Glu519 and Glu521 to Lys in C-terminal tail altered the pH dependence of Na(+)/H(+) and Li(+)/H(+) exchange activities. These results indicate that the specific acidic amino acid residues at C-terminal domain play important roles for the K(m) and the pH dependence of the exchange activity.     

Acid-base catalysis in the chemical mechanism of inosine monophosphate dehydrogenase

Inosine-5'-monophosphate dehydrogenase (IMPDH) catalyzes the K+-dependent reaction IMP + NAD + H2O --> XMP + NADH + H+ which is the rate-limiting step in guanine nucleotide biosynthesis. The catalytic mechanism of the human type-II IMPDH isozyme has been studied by measurement of the pH dependencies of the normal reaction, of the hydrolysis of 2-chloro-IMP (which yields XMP and Cl- in the absence of NAD), and of inactivation by the affinity label 6-chloro-purine-ribotide (6-Cl-PRT). The pH dependence of the IMPDH reaction shows bell-shaped profiles for kcat and the kcat/Km values for both IMP and NAD, illustrating the involvement of both acidic and basic groups in catalysis. Half-maximal kcat values occur at pH values of 7.2 and 9.8; similar pK values of 6.9 and 9.4 are seen in the kcat/Km profile for NAD. The kcat/Km profile for IMP, which binds first in the predominantly ordered kinetic mechanism, shows pK values of 8.1 and 7.3 for acidic and basic groups, respectively. None of the kinetic pK values correspond to ionizations of the free substrates and thus reflect ionization of the enzyme or enzyme-substrate complexes. The rate of inactivation by 6-Cl-PRT, which modifies the active site sulfhydryl of cysteine-331, increases with pH; the pK of 7.5 reflects the ionization of the sulfhydryl in the E.6-Cl-PRT complex. The pKs of the acids observed in the IMPDH reaction likely also reflect ionization of the cysteine-331 sulfhydryl which adds to C-2 of IMP prior to NAD reduction. The kcat and kcat/Km values for hydrolysis of 2-Cl-IMP show a pK value of 9.9 for a basic group, similar to that seen in the overall reaction, but do not exhibit the ionization of an acidic group. Surprisingly, the rates of 2-Cl-IMP hydrolysis and of inactivation by 6-Cl-PRT are not stimulated by K+, in contrast to the >100-fold K+ activation of the IMPDH reaction. Apparently the enigmatic role of K+ lies in the NAD(H)-dependent segment of the IMPDH reaction. To evaluate the importance of hydrogen bonding in substrate binding, several deamino- and deoxy-analogues of IMP were tested as substrates and inhibitors. Only 2'-deoxy-IMP was a substrate; the other compounds tested were competitive inhibitors with Ki values at most 10-fold greater than the KD for IMP, illustrating the greater importance of hydrogen-bonding interactions in the chemistry of the IMPDH reaction than simply in nucleotide binding.     

A catalytically deficient active site variant of PvuII endonuclease binds Mg(II) ions

In efforts to understand the mechanisms of many nucleic acid enzymes, the first site-directed mutations are made at conserved acidic active residues. Almost without exception, the low or null activities of the resulting variants are attributed to the importance of the acidic residue(s) to the ligation of required metal ions. Using (25)Mg NMR spectroscopy as a direct probe of metal ion binding and the homodimeric PvuII restriction endonuclease as a model system, this interpretation is examined and clarified. Our results indicate that Mg(II) binds wild-type PvuII endonuclease in the absence of DNA with a K(d,app) of 1.9 mM. Hill analysis yields an n(H) coefficient of 1.4, a value consistent with the binding of more than one Mg(II) ion per monomer active site. Variable pH studies indicate that two ionizable groups are responsible for Mg(II) binding by wild-type PvuII endonuclease near physiological pH. The pK(a,app) for these ionizations is 6.7, a value which is unusual for acidic residues but consistent with data obtained for critical groups in MunI endonuclease and a number of other hydrolases. To assign residues critical to ligating Mg(II), binding measurements were performed on the low activity catalytic site mutants E68A and D58A. As expected, E68A binds Mg(II) ions very weakly (K(d,app) approximately 40 mM), implicating Glu68 as critical to Mg(II) binding. Interestingly, while D58A has only residual specific activity, it retains an affinity for Mg(II) with a K(d,app) of 3.6 mM and exhibits a Hill coefficient of 0.7. Moreover, in this variant, multiple ionizable groups with pK(a,app) of 7.2 are involved in Mg(II) binding, suggesting a shuffling of Mg(II) ligands in the active site. These data indicate that Asp58 is important for the critical positioning of metal ion(s) required for catalysis.     

pK values of histidine residues in ribonuclease Sa: effect of salt and net charge

The primary goal of this study was to gain a better understanding of the effect of environment and ionic strength on the pK values of histidine residues in proteins. The salt-dependence of pK values for two histidine residues in ribonuclease Sa (RNase Sa) (pI=3.5) and a variant in which five acidic amino acids have been changed to lysine (5K) (pI=10.2) was measured and compared to pK values of model histidine-containing peptides. The pK of His53 is elevated by two pH units (pK=8.61) in RNase Sa and by nearly one pH unit (pK=7.39) in 5K at low salt relative to the pK of histidine in the model peptides (pK=6.6). The pK for His53 remains elevated in 1.5M NaCl (pK=7.89). The elevated pK for His53 is a result of screenable electrostatic interactions, particularly with Glu74, and a non-screenable hydrogen bond interaction with water. The pK of His85 in RNase Sa and 5K is slightly below the model pK at low salt and merges with this value at 1.5M NaCl. The pK of His85 reflects mainly effects of long-range Coulombic interactions that are screenable by salt. The tautomeric states of the neutral histidine residues are changed by charge reversal. The histidine pK values in RNase Sa are always higher than the pK values in the 5K variant. These results emphasize that the net charge of the protein influences the pK values of the histidine residues. Structure-based pK calculations capture the salt-dependence relatively well but are unable to predict absolute histidine pK values.     

Fourier transform infrared evidence for early deprotonation of Asp(85) at alkaline pH in the photocycle of bacteriorhodopsin mutants containing E194Q

The role of the extracellular Glu side chains of bacteriorhodopsin in the proton transport mechanism has been studied using the single mutants E9Q, E74Q, E194Q, and E204Q; the triple mutant E9Q/E194Q/E204Q; and the quadruple mutant E9Q/E74Q/E194Q/E204Q. Steady-state difference and deconvoluted Fourier transform infrared spectroscopy has been applied to analyze the M- and N-like intermediates in membrane films maintained at a controlled humidity, at 243 and 277 K at alkaline pH. The mutants E9Q and E74Q gave spectra similar to that of wild type, whereas E194Q, E9Q/E194Q/E204Q, and E9Q/E74Q/E194Q/E204Q showed at 277 K a N-like intermediate with a single negative peak at 1742 cm(-1), indicating that Asp(85) and Asp(96) are deprotonated. Under the same conditions E204Q showed a positive peak at 1762 cm(-1) and a negative peak at 1742 cm(-1), revealing the presence of protonated Asp(85) (in an M intermediate environment) and deprotonated Asp(96). These results indicate that in E194Q-containing mutants, the second increase in the Asp(85) pK(a) is inhibited because of lack of deprotonation of the proton release group. Our data suggest that Glu(194) is the group that controls the pK(a) of Asp(85).