Correlation of low-barrier hydrogen bonding and oxyanion binding in transition state analogue complexes of chymotrypsin

The structures of the hemiketal adducts of Ser 195 in chymotrypsin with N-acetyl-L-leucyl-L-phenylalanyl trifluoromethyl ketone (AcLF-CF3) and N-acetyl-L-phenylalanyl trifluoromethyl ketone (AcF-CF3) were determined to 1.4-1.5 A by X-ray crystallography. The structures confirm those previously reported at 1.8-2.1 A [Brady, K., Wei, A., Ringe, D., and Abeles, R. H. (1990) Biochemistry 29, 7600-7607]. The 2.6 A spacings between Ndelta1 of His 57 and Odelta1 of Asp 102 are confirmed at 1.3 A resolution, consistent with the low-barrier hydrogen bonds (LBHBs) between His 57 and Asp 102 postulated on the basis of spectroscopy and deuterium isotope effects. The X-ray crystal structure of the hemiacetal adduct between Ser 195 of chymotrypsin and N-acetyl-L-leucyl-L-phenylalanal (AcLF-CHO) has also been determined at pH 7.0. The structure is similar to the AcLF-CF3 adduct, except for the presence of two epimeric adducts in the R- and S-configurations at the hemiacetal carbons. In the (R)-hemiacetal, oxygen is hydrogen bonded to His 57, not the oxyanion site. On the basis of the downfield 1H NMR spectrum in solution, His 57 is not protonated at Nepsilon2, and there is no LBHB at pH >7.0. Because addition of AcLF-CHO to chymotrypsin neither releases nor takes up a proton from solution, it is concluded that the hemiacetal oxygen of the chymotrypsin-AcLF-CHO complex is a hydroxyl group and not attracted to the oxyanion site. The protonation states of the hemiacetal and His 57 are explained by the high basicity of the hemiacetal oxygen (pK(a) > 13.5) relative to that of His 57. The 13C NMR signal for the adduct of AcLF-13CHO with chymotrypsin is consistent with a neutral hemiacetal between pH 7 and 13. At pH <7.0, His 57 in the AcLF-CHO-hemiacetal complex of chymotrypsin undergoes protonation at Nepsilon2 of His 57, leading to a transition of the 15.1 ppm downfield signal to 17.8 ppm. The pK(a)s in the active sites of the AcLF-CF3 and AcLF-CHO adducts suggest an energy barrier of 6-7 kcal x mol(-1) against ionizations that change the electrostatic charge at the active site. However, ionizations of neutral His 57 in the AcLF-CHO-chymotrypsin adduct, or in free chymotrypsin, proceed with no apparent barrier. Protonation of His 57 is accompanied by LBHB formation, suggesting that stabilization by the LBHB overcomes the barrier to ionization. On the basis of the hydration constant for AcLF-13CHO and its inhibition constant, its K(d) is 16 microM, 8000-fold larger than the comparable value for AcLF-CF3.     

Mechanism of slow-binding inhibition of human leukocyte elastase by trifluoromethyl ketones

Kinetics of inhibition have been determined for the interaction of human leukocyte elastase (HLE) with two series of peptide trifluoromethyl ketones (TFMKs): X-Val-CF3,X-Pro-Val-CF3,X-Val-Pro-Val-CF3, and X-Lys(Z)-Val-Pro-Val-CF3, where X is MeOSuc or Z. These compounds are "slow-binding" inhibitors of HLE and, thus, allow the determination of Ki, the dissociation constant for the stable complex of inhibitor and enzyme, as well as kon and koff, the rate constants for formation and decomposition of this complex. Maximal potency is reached with Z-Lys(Z)-Val-Pro-Val-CF3, which displays a Ki less than 0.1 nM. Upon binding to HLE, these compounds undergo addition by the hydroxyl of the active site serine to form a hemiketal. The evidence supporting a hemiketal intermediate includes Ki values of 1.6 and 80,000 nM for Z-Val-Pro-Val-CF3 and its alcohol analogue, linear free energy correlations between inhibitory potency and catalytic efficiency for structurally related TFMKs and substrates, and the pH dependence of kon for the inhibition of HLE by Z-Val-Pro-Val-CF3, which is sigmoidal and displays a pKa of 6.9. Hemiketal formation is probably not rate limiting, however. Kinetic solvent isotope effects of unity suggest that kon cannot be rate limited by a reaction step, like hemiketal formation, that is subject to protolytic catalysis. A general mechanism that is consistent with these results is one in which formation of the hemiketal is rapid and is followed or preceded by a slow step that rate limits kon.(ABSTRACT TRUNCATED AT 250 WORDS)     

Specificity and stability of the chymotrypsin inhibitor from winged bean seed (Psophocarpus tetragonolobus (L) Dc)

The specificity of the winged bean chymotrypsin inhibitor is restricted to the chymotrypsins (EC 3.4.21.1 and EC 3.4.21.2). Trypsins (EC 3.4.21.4), elastase (EC 3.4.21.11), subtilisins (EC 3.4.21.14), proteinase K (EC 3.4.21.14) and Pronase (EC 3.4.24.4) are not inhibited. The inhibitor reacts with two molecules of chymotrypsin to form a stable complex (Mr approx. 70 0000) which was isolated by gel filtration on Sephadex G-100. When mixed with substrate, the interaction of the inhibitor with alpha-chymotrypsin is characterized by substrate-induced dissociation of the complex. In contrast, the interaction with chymotrypsin B is quantitative with no substrate-induced dissociation. The inhibitor reacts with alpha-chymotrypsin to form a 1 : 2 molar complex at all ratios of [I]/[E]; however, the interaction with chymotrypsin B is characterized by the formation of initially of a 1 : 1 molar complex at [I] greater than [E] followed by the formation of the 1 : 2 molar complex at [I] less than 2[E]; an intermediate species of Mr approx. 48 000 was demonstrated by gel filtration on Sephadex G-100. The inhibitor is stable over the pH range 2.0-11.5 and to heating up to 70 degrees C at pH 4.1 and 8.0, and up to 90 degrees C at pH 3.0. The inhibitor resists denaturation in 8.0 M urea at pH 8.0 and 4.0, is stable in 0.12 M beta-mercaptoethanol at pH 8.0; however, reduction in 8.0 M urea results in a loss of inhibitory activity. The inhibitor resists digestion with pepsin at pH 2.0, being only slowly degraded over a period of 7 days with an equimolar amount of pepsin.     

Porcine chymotrypsin A-pi, a more acidic chymotrypsin.

A kinetic study of procine chymotrypsin A-pi revealed two characteristic properties of this type of chymotrypsin: 1. Porcine chymotrypsin A-pi, like bovine chymotrypsin B-pi does not bind proflavin, which is a competitive inhibitor of bovine trypsin and chymotrypsin A-alpha. 2. The pH profiles of the steady-state parameters show the two usual important pK's. The basic one, pK2 = 9.6, affects both Km and kcat/Km and probably controls the binding conformation of chymotrypsin. The acidic one, pK1 = 5.7, affects kcat and kcat/Km and plays a role in the catalytic process. The value of pK1 is unusually low.     

Turkey ovomucoid third domain inhibits eight different serine proteinases of varied specificity on the same ...Leu18-Glu19 ... reactive site

We show that eight different serine proteinases--bovine chymotrypsins A and B, porcine pancreatic elastase I, proteinase K, Streptomyces griseus proteinases A and B, and subtilisins BPN' and Carlsberg--interact with turkey ovomucoid third domain at the same Leu18-Glu19 peptide bond, the reactive site of the inhibitor. Turkey ovomucoid third domain was converted to modified (the reactive site peptide bond hydrolyzed) form as documented by sequencing. Complexes of all eight enzymes both with virgin and with modified inhibitor were prepared. All 16 complexes were subjected to kinetically controlled dissociation, and all 16 produced predominantly virgin (greater than 90%) inhibitor, thus proving our point. During this investigation, we found that both alpha-chymotrypsin and especially S. griseus proteinase B convert virgin to modified turkey ovomucoid third domain, even in the pH range 1-2, a much lower pH than we expected. We have also measured rate constants kon and kon* for the association of virgin and modified turkey ovomucoid third domain with several serine proteinases. The kon/kon* ratio is 4.8 X 10(6) for chymotrypsin, but it is only 1.5 for subtilisin Carlsberg. A number of generalizations concerning reactive sites of protein proteinase inhibitor are proposed and discussed.     

pH dependence of the four individual transitions in the catalytic S-cycle during photosynthetic oxygen evolution

We have investigated the pH dependence for each individual redox transition in the S-cycle of the oxygen evolving complex (OEC) of photosystem II by electron paramagnetic resonance (EPR) spectroscopy. In the experiments, OEC is advanced to the appropriate S-state at normal pH. Then, the pH is rapidly changed, and a new flash is given. The ability to advance to the next S-state in the cycle at different pHs is determined by measurements of the decrease or increase of characteristic EPR signals from the OEC in different S-states. In some cases the measured EPR signals are very small (this holds especially for the S0 ML signal at pH >7.5 and pH <4.8). Therefore, we refrain from providing error limits for the determined pK's. Our results indicate that the S1 --> S2 transition is independent of pH between 4.1 and 8.4. All other S-transitions are blocked at low pH. In the acidic region, the pK's for the inhibition of the S2 --> S3, the S3 --> [S4] --> S0, and the S0 --> S1 transitions are about 4.0, 4.5, and 4.7, respectively. The similarity of these pK values indicates that the inhibition of the steady-state oxygen evolution in the acidic range, which occurs with pK approximately 4.8, is a consequence of similar pH blocks in three of the redox steps involved in the oxygen evolution. In the alkaline region, we report a clear pH block in the S3 --> [S4] --> S0 transition with a pK of about 8.0. Our study also indicates the existence of a pH block at very high pH (pK approximately 9.4) in the S2 --> S3 transition. The S0 --> S1 transition is not affected, at least up to pH 9.0. This suggests that the inhibition of the steady-state oxygen evolution, which occurs with a pK of 8.0, is dominated by the inhibition of the S3 --> [S4] --> S0 transition. Our results are obtained in the presence of 5% methanol (v/v). However, it is unlikely that the determined pK's are affected by the presence of methanol since our results also show that the pH dependence of the steady-state oxygen evolution is not affected by methanol. The results in the alkaline region are in good agreement with a model, which suggests that the redox potential of Y(Z*)/Y(Z) is directly affected by high pH. At high pH the Y(Z*)/Y(Z) potential becomes lower than that of S2/S1 and S3/S2. The acidic block, with a pK of 4-5 in three S-transitions, implies that the inhibition mechanism is similar, and we suggest that it reflects protonation of a carboxylic side chain in the proton relay that expels protons from the OEC.     

High resolution nuclear magnetic resonance studies of the active site of chymotrypsin. II. Polarization of histidine 57 by substrate analogues and competitive inhibitors

The proton nuclear magnetic resonance signal of the His57-Asp102 hydrogen bonded proton in the charge relay system of chymotrypsinogen A and chymotrypsin A_δ has been monitored to determine the influence of substrate analogues and competitive inhibitors on the electronic state of the active site regions. Borate ion, benzene boronic acid and 2-phenylethylboronic acid, when bound to chymotrypsin at pH 9.5 shift the resonance position of the His-Asp hydrogen bonded proton to ?15.9, ?16.3 and ?17.2 parts per million, respectively. These positions are intermediate between the low pH position in the free enzyme of ?18.0 parts per million and the high pH position of ?14.9 parts per million. The presence of these analogues prevents the His-Asp proton resonance from titrating in the region of pH 6 to 9.5. Similar low field shifts are observed for the hydrogen bonded proton resonance of subtilisin BPN′ when complexed with these boronic acids. The results support the chemical and crystallographic data which show that negatively charged tetrahedral adducts of the boronic acid substrate analogues are formed at the active sites of these enzymes. When combined with similar nuclear magnetic resonance data for the binding of N-acetyl-l-tryptophan to chymotrypsin A_δ, they suggest that a direct interaction occurs between the active site histidine and the atom occupying the leaving group position of the substrate, presumably a hydrogen bond.The His-Asp proton resonance was also monitored in complexes of chymotrypsin A_δ with bovine pancreatic trypsin inhibitor over the pH range 4 to 9. In the complex the low field proton resonance had a field position of ?14.9 parts per million over the pH range 4 to 9 indicating that His57 is in the neutral form, similar to the active enzyme at high pH.     

Nucleotide binding to Na,K-ATPase: pK values of the groups affecting the high affinity site

Investigation of the ionic strength effect on the interactions between nucleotides (ATP and ADP) and Na,K-ATPase in a broad pH range was aimed at revealing pK values of the charged groups of the interacting species. Ionic strength experiments suggested that an amino acid residue with a pK > 8.0 is part of the protein binding site. A combination of equilibrium and transient experiments at various pH values allowed for the characterization of the groups electrostatically involved in either the association process (kon) or the stability of the preformed complexes (koff). Two groups (pK1 = 6.7 and pK2 = 8.4) appear to be important for the proper organization of the binding site and, therefore, the association reaction. Moreover, deprotonation of the basic group completely precludes association. pH dependencies of the dissociation rate constants for ATP and ADP are very different. An increase in pH from 5 to 9.5 induces a 9-fold increase in koff for ATP, whereas koff for ADP decreases 4-fold between pH 5 and 8, and decreases further in the alkaline region. A comparison of the pH dependencies for koff for ATP and ADP suggests two effects: (1) at acidic pH, the value of the total negative charge of the nucleotide determines the tightness of binding; and (2) short-range interactions involving the terminal phosphate group are important for nucleotide dissociation from the site. The difference in the pH dependencies of koff for the nucleotides suggests the existence of positive charges in close proximity to Asp369, relieving the repulsion between the gamma-phosphate of ATP and Asp369.     

Inhibition kinetics of acetylcholinesterase with fluoromethyl ketones

A series of trifluoromethyl ketones that reversibly inhibit acetylcholinesterase and pseudocholinesterase were synthesized. By analogy to chymotrypsin and on the basis of data reported here, we propose that the active-site serine adds to the ketone to form an ionized hemiketal. The compound (5,5,5-trifluoro-4-oxopentyl)trimethylammonium bicarbonate (1) inhibits acetylcholinesterase with Ki = 0.06 X 10(-9)M and pseudocholinesterase with Ki = 70 X 10(-9)M. Replacement of the nitrogen of 1 by carbon (compound 2) increases Ki for 1 200-fold for acetylcholinesterase but does not significantly alter Ki for pseudocholinesterase. The Ki for the methyl ketone corresponding to 2 is 2 X 10(-4)M for both enzymes, as compared with 12 X 10(-9)M for the trifluoromethyl ketone (acetylcholinesterase). For both enzymes, a linear decrease in log Ki with decreasing pK of the inhibitor hydrate was observed with ketones containing from 0 to 3 fluorines. We attribute this effect to the stabilization of the hemiketal oxyanion. The reduction of the pK of the hemiketal by the trifluoromethyl group is an important contributing factor to the low Ki of trifluoromethyl ketones. The inhibition of acetylcholinesterase by tetramethylammonium chloride and trifluoroacetone was compared to the inhibition by 1, which is a composite of the two smaller inhibitors. The entropic advantage of combining the smaller inhibitors into one molecule is 1.1 X 10(3)M. Inhibitors with Ki less than or equal to 70 X 10(-9) M are slow binding (Morrison, 1982; Morrison & Walsh, 1988). The kinetic data do not require formation of a noncovalent complex prior to formation of the ketal, although such a complex(es) cannot be excluded.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reactivity of small thiolate anions and cysteine-25 in papain toward methyl methanethiosulfonate

The dependence on thiol pK of the second-order rate constant (kS) for reaction of thiolate anions with MMTS was shown to follow the Br?nsted equation log kS = log G + beta pK with log G = 1.44 and 3.54 and beta = 0.635 and 0.309 for aryl and alkyl thiols, respectively. The reactivity toward MMTS of the protonated thiol group was found to be negligible in comparison to that of the thiolate anion. For 2-mercaptoethanol the reactivity toward MMTS of the protonated form of the thiol group was shown to be at least 5 X 10(9) smaller than that of the thiolate anion. The pH dependence of the second-order rate constant for reaction of the thiolate group of Cys-25 at the active site of papain was determined and shown to be consistent with the previously determined low pK for Cys-25 and its electrostatic interaction with His-159. The small dependence of the reactivity of Cys-25 on thiol pK (beta approximately 0.09) suggested that the charge-charge interactions that act through space to perturb the pK of the nucleophile at the active site of papain and perhaps other enzymes may serve to increase the fraction of nucleophile present in the reactive basic form without introducing the decrease in nucleophilic reactivity seen in model systems where pK's are lowered primarily by charge-dipole interactions.     

Ionization of amino acid residues involved in the catalytic mechanism of aspartate transcarbamoylase.

The chemical and kinetic mechanisms of the reaction catalyzed by the catalytic trimer of aspartate transcarbamoylase have been examined. The variation of the kinetic parameters with pH indicated that at least four ionizing amino acid residues are involved in substrate binding and catalysis. The pH dependence of K(ia) for carbamoyl phosphate and the K(i) for N-(phosphonoacetyl)-L- aspartate revealed that a protonated residue with a pK value of 9.0 is required for the binding of carbamoyl phosphate. However, the variation with pH of K(i) for succinate, a competitive inhibitor of aspartate, and for cysteine sulfinate, a slow substrate, showed that a single residue with a pK value of 7.3 must be protonated for binding these analogues and, by inference, aspartate. The profile of log V against pH displayed a decrease in reaction rate at low and high pH, suggesting that two groups associated with the Michaelis complex, a deprotonated residue with a pK value of 7.2 and a protonated group with a pK value of 9.5, are involved in catalysis. By contrast, the catalytically productive form of the enzyme-carbamoyl phosphate complex, as illustrated in the bell-shaped pH dependence of log (V/K)(asp), is one in which a residue with a pK value of 7.0 must be protonated while a group with a pK value of 9.1 is deprotonated. This interpretation is supported by the results from the temperature dependence of the V and V/K profiles and from the pH dependence of pK(i) for the aspartate analogues.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pigeon liver diacetyl reductase. Effects of pH on the kinetic parameters of the reaction.

(1) The pH dependence of the kinetic parameters of the reaction catalyzed by pigeon liver diacetyl reductase (EC 1.1.1.5) was investigated in the pH range 5.1-8.6. (2) From the results obtained it is postulated that: (a), a group of pK around 7, active in the protonated form, participates in the interaction of the enzyme with NADH and NAD. (b), a second group with a pK of 8.4, active in the protonated form too, takes part in the binding of diacetyl to E-NADH. (c) A third group of pK about 4.7-5, active in the unprotonated form, is involved at least in the dissociation of the complex E-NAD and in the attachment of diacetyl to E-NADH.     

Regulation of the 2-oxoglutarate dehydrogenase lipoate succinyltransferase complex from cauliflower by nucleotide. Pre-steady state kinetics and physical studies

Analysis of the binding of thiamin pyrophosphate to the 2-oxoglutarate dehydrogenaselipoate succinyltransferase multienzyme complex using pre-steady state kinetic methods revealed that the presence of 2-oxoglutarate is not necessary for binding, although it does stabilize the complex by slowing the rate of dissociation of the holoenzyme. The rate of binding of thiamin-PPi to the enzyme and the subsequent enzyme activation are not limited by a reaction at C-2 of the thiazolium ring of thiamin-PPi since no kinetic isotope effect is observed when 2-D-thiamin-PPi is substituted for the protonated cofactor. The presence of 5'-AMP, which activates the reaction producing both a V and a Km response, causes a significant increase in kon for thiamin-PPi. The AMP analog 1,N6-ethenoadenosine-5'-monophosphate (epsilon-AMP) also activates the reaction, but shows only a K effect, with no influence on V. This effector reduces Kd for the thiamin-PPi2-oxoglutarate dehydrogenase complex by increasing kon. The change in kon for thiamin-PPi in response to changes in hydrogen ion concentration shows pK values which are unaffected by the addition of AMP, in this respect resembling the steady state kinetic response of V/Km and differing from the pH profile of V. The dissociation constant of holoenzyme is relatively insensitive to pH over the range pH 6 to 9, but in the presence of AMP the Kd, which is decreased in the range from pH 7 to 8, increases sharply at higher or lower pH values.     

Temperature- and pH-dependent changes in the coordination sphere of the heme c group in the model peroxidase N alpha-acetyl microperoxidase-8.

The pH- and temperature-dependent changes in the coordination sphere of the heme c group of N alpha-acetyl microperoxidase-8 (Ac-MP-8) have been studied by examining its optical, resonance Raman, electron paramagnetic resonance, and magnetic circular dichroism spectra. An optical titration indicates that Ac-MP-8 exists in three major ionization forms over the pH 1-12 range that are linked by pK alpha values of approximately 3 and 9. The acid form that is present at pH 1.5 exists as a mixture of five- and six-coordinate high-spin species and most likely has water or buffer ions as axial ligand(s). On titration to pH 7, the His18 residue is deprotonated and becomes the proximal ligand to the iron to give a six-coordinate neutral form that has water as the sixth ligand. This form exists in a thermal high-spin intermediate-spin state equilibrium. On raising the pH to 10, an alkaline form is generated which is predominantly a five-coordinate high-spin species. It is formed by ionization of the proximal His18 residue to its imidazolate form with concomitant dissociation of the water ligand at the sixth site. At concentrations of Ac-MP-8 greater than 10 microM, some six-coordinate low-spin species are formed that are attributed to a dimer in which a His18 residue from a second molecule of Ac-MP-8 coordinates to the sixth site of another to give a bis-His complex. Raising the pH to 11.5 does not produce an appreciable amount of the six-coordinate complex with hydroxide as the sixth ligand. These studies show that Ac-MP-8 is a good water-soluble model for the peroxidases that exhibits minimal aggregation at concentrations below 10 microM in the neutral and alkaline pH regions.     

Salt effects on ionization equilibria of histidines in myoglobin

The salt dependence of histidine pK(a) values in sperm whale and horse myoglobin and in histidine-containing peptides was measured by (1)H-NMR spectroscopy. Structure-based pK(a) calculations were performed with continuum methods to test their ability to capture the effects of solution conditions on pK(a) values. The measured pK(a) of most histidines, whether in the protein or in model compounds, increased by 0.3 pH units or more between 0.02 M and 1.5 M NaCl. In myoglobin two histidines (His(48) and His(36)) exhibited a shallower dependence than the average, and one (His(113)) showed a steeper dependence. The (1)H-NMR data suggested that the salt dependence of histidine pK(a) values in the protein was determined primarily by the preferential stabilization of the charged form of histidine with increasing salt concentrations rather than by screening of electrostatic interactions. The magnitude and salt dependence of interactions between ionizable groups were exaggerated in pK(a) calculations with the finite-difference Poisson-Boltzmann method applied to a static structure, even when the protein interior was treated with arbitrarily high dielectric constants. Improvements in continuum methods for calculating salt effects on pK(a) values will require explicit consideration of the salt dependence of model compound pK(a) values used for reference in the calculations.     

H NMR investigation of the influence of interacting sites on the dynamics and thermodynamics of substrate and ligand binding to horseradish peroxidase.

The influence of substrate benzhydroxamic acid (BHA) and iron ligand (cyanide) on the thermodynamics and dynamics of each of the two binding sites of horseradish peroxidase (HRP) isozyme C has been investigated by 1H NMR spectroscopy. A combination of line-width analysis and saturation transfer spectroscopy has allowed the direct determination of the off-rate of substrate and ligand in the absence or presence of the other. These off-rates, together with available dissociation constants obtained by optical spectroscopy (Schonbaum, 1973), provide estimates for kon. The dissociation constant for cyanide binding to the BHA.HRP complex was also directly determined by NMR. In all cases the 1H NMR determined dynamic and thermodynamic data agree well with those values available in the literature. BHA binding leads to a 200-fold decrease in CN- affinity that arises from a factor greater than 10 decrease in koff(CN-) and greater than 2 x 10(3) decrease in kon(CN-). While a portion of the decrease in kon(CN-) can be rationalized by water coordination of the iron in the BHA.HRP complex, the additional decrease in kon(CN-) and that in koff(CN-) indicates that BHA in the binding pocket blocks the CN- ligation channel and serves as a "gate" to CN- exchange. This view is supported by observing a factor greater than 4 decrease in distal His labile proton exchange with bulk water in HRP-CN upon BHA binding. The ternary complex BHA.HRP-CN is shown to be heterogeneous. While the thermodynamics of BHA and CN- binding appear similar in the two ternary complexes, the BHA on- and off-rates for the two complexes differ by a factor of approximately 10. The two heterogeneous forms interconvert at 25 degrees C at approximately 2 x 10(2) s-1, precluding the determination of any difference in the CN- binding rates by saturation transfer. The greater lability of one of the two ternary complexes is attributed to an alternate orientation of some distal residue that blocks the substrate binding channel in one of the forms. Transferred nuclear Overhauser effects from the heme to BHA in the ternary complex reveal that the BHA substrate is in contact not only with the heme pyrrole D substituents but also with the distal His 42, indicating that the polar side chain of BHA extends well into the distal heme pocket.(ABSTRACT TRUNCATED AT 400 WORDS)     

Phosphorylated aminosugars: synthesis, properties, and reactivity in enzymatic reactions

A number of phosphorylated aminosugars have been prepared and tested as substrates for metabolic reactions. 6-Aminoglucose is a slow substrate for yeast hexokinase with a Vmax that is only 0.012% that for glucose. While Vmax is pH independent, V/K decreases below the pK of 9.0 of the amino group. 6-Aminoglucose is a competitive inhibitor vs glucose with a Ki value increasing below the pK of 9 but leveling off at 33 mM below pH 7.16. Thus, protonation decreases binding affinity by 2.4 kcal/mol and only the neutral amine is catalytically competent. 6-Aminoglucose-6-P was synthesized enzymatically with hexokinase. Its pK's determined by 31P NMR were 2.46 and 8.02 (alpha anomer) and 2.34 and 7.85 (beta anomer), with a beta:alpha ratio of 3.0. It is most stable at pH 12 (half-life 228 h at 22 degrees C), while as a monoanion its half-life is 3 h. The free energy of hydrolysis at 25 degrees C and pH 9.25 is -10.3 kcal/mol. The phosphorylated amino analogues of 6-P-gluconate, ribulose-5-P, fructose-6-P, fructose-1,6-bis-P (amino group at C-6 only), and glyceraldehyde-3-P were synthesized enzymatically. The 31P NMR chemical shifts of these analogues are 8-8.5 ppm at pH 9.5. Their relative stability is 6-aminogluconate-6-P greater than 3-aminoglyceraldehyde-3-P greater than 6-aminoglucose-6-P greater than 6-aminofructose-1,6-bis-P congruent to 6-aminofructose-6-P greater than 5-aminoribulose-5-P. These analogues were tested as substrates for their respective enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)     

The pH dependence of exchange transport of glucose in human erythrocytes

In glucose exchange transport into red blood cells the rate of glucose uptake showed two pH dependent maxima, with the larger at approximately pH 7.5 and the smaller one at pH 3. In the studied pH range the relation between the rate of glucose uptake and the substrate concentration followed Michaelis-Menten kinetics. While the maximal velocity (V) reflected the pH changes of the media, the Michaelis constant (K_m) remained constant. The dissociation constants of the groups of the free carrier and the carrier-glucose complex were the same. The pK of the acidic group was 5.2 and of the basic group 9.5. Glucose was not bound to groups of the carrier which dissociated protons in the pH range of three to nine. By rearranging the equation for the pH dependence of the relative influx a more definitive graphic determination of the pK values was produced.     

The AdhS alleloenzyme of alcohol dehydrogenase from Drosophila melanogaster. Variation of kinetic parameters with pH.

The variation with pH of the kinetic parameters for the alcohol and acetaldehyde reactions were studied for the alleloenzyme AdhS from Drosophila melanogaster. The variation of Ki (KEO,I) with pH for two ethanol-competitive inhibitors, pyrazole and 2,2,2-trifluoroethanol, was also studied. Both alcohol oxidation and acetaldehyde reduction follow a compulsory ordered pathway, with coenzyme binding first. The rate-limiting step for ethanol oxidation is complex and involves at least hydride transfer and dissociation of the enzyme-NADH complex (ER). In contrast with this, the rate-limiting step for the back reaction, i.e. the reduction of acetaldehyde, is dissociation of the enzyme-NAD+ complex (EO). A rate-limiting ER dissociation appears in the oxidation of the secondary alcohol propan-2-ol, whereas for the back reaction, i.e. acetone reduction, hydride transfer in the ternary complexes is rate-limiting. There is one group in the free enzyme, with a pK of approx. 8.0, that regulates the kon velocity for NADH, whereas for NAD+ several groups seem to be involved. A group in the enzyme is drastically perturbed by the formation of the binary EO complex. Protonation of this group with a pK of 7.6 in the EO complex resulted in weakened alcohol and inhibitor binding, in addition to an increased dissociation rate of NAD+ from the binary EO complex. Neither the binding of acetaldehyde nor the dissociation rate of NADH from the binary ER complex varied within the pH region studied.     

pH dependence of progesterone interaction with progesterone-binding globulin. Kinetic and equilibrium studies.

The kinetics of binding and dissociation for the progesterone-binding globulin (PBG)-progesterone complex have been measured as a function of pH. The association rate constant appears to be independent of pH from pH to 10 with an average value of kon = 8.5 X 10(7)M-1 S-1. The dissociation rate constant is strongly pH dependent with the dependency defined by: koff = k0 (1 + [H+]/K1 + K2/[H+])(1 + K3*/[H+])/(1 + K3/[H+]). The best values for the various parameters were k0 = 0.0785 s-1, pK1 = 5.30, pK2 = 10.54, pK3* = 7.41, and pK3 = 7.21. Simpler expressions were inadequate to fit the data, and it was concluded that at least three ionizing residues are responsible for the stability of the PBG-progesterone complex. The affinity constant was determined by equilibrium dialysis over the range of pH 3 to 12. The ratio of the association and dissociation rate constants is in agreement with the affinity constant from pH 6.5 to 10.5. The influence of pH on the conformation and binding activity of PBG was also investigated. Denaturation by acid, base, or guanidine hydrochloride leads to a reversible loss of binding activity. Regain of binding activity in all cases is slow with half-times of 0.5 to 2.7 h, depending on conditions. The rate of acid denaturation was found to be incompletely protonated at pH 1.4, suggesting a buried carboxylic acid residue. The slow renaturation of PBG might be due to the difficulty of burying a charged residue in the protein's interior coupled with steric hindrance by the large carbohydrate moiety of PBG.