Active site generation of a protonically unstable suicide substrate from a stable precursor: glucose oxidase and dibromonitromethane.

Bromonitromethane is an inefficient suicide substrate for glucose oxidase (in contrast to the case of CH(3)CCl=NO(2)(-) and D-amino acid oxidase) because, in the enzyme-substrate encounter step, the required ionization states of enzyme (EH(0)(+), pK(a) approximately 3.5) and substrate (CHBr=NO(2)(-), pK(a) approximately 8.3) cannot be highly populated simultaneously. Because reprotonation of CHBr=NO(2)(-) is rapid at the pH value used for the assay of glucose oxidase, presentation of the enzyme with the preformed anion could not be exploited in this case. We circumvent this difficulty by allowing the enzyme to reductively dehalogenate CHBr(2)NO(2), thereby generating the desired protonically unstable suicide substrate in situ (E(r) + CHBr(2)NO(2) --> E(o) + CHBr=NO(2)(-) + HBr + H(+)). Irreversible inactivation of the enzyme, because of the formation of a dead-end N-5 formylflavin adduct, is more than 100-fold faster when CHBr=NO(2)(-) is generated in situ than when it is externally applied. The remaining competitive fates of CHBr=NO(2)(-) at the active site are protonation and release or oxidation to HCOBr (or HCONO(2)). Strong support for these conclusions comes from (1) the brisk evolution of CH(3)CBr=NO(2)(-) (which is too bulky to act further as an efficient suicide substrate) from the enzyme-catalyzed reductive debromination of CH(3)CBr(2)NO(2), (2) the 1:1 stoichiometry of enzyme inactivation, and (3) the identification of the modified flavin as 5-formyl-1, 5-dihydro-FAD.

Linear free energy relationships implicate three modes of binding for fluoroaromatic inhibitors to a mutant of carbonic anhydrase II.

[figure: see text] Linear free energy relationships between binding affinity and hydrophobicity for a library of fluoroaromatic inhibitors of F131V carbonic anhydrase II (CA) implicate three modes of interaction. X-ray crystal structures suggest that F131 interacts with fluoroaromatic inhibitors, while P202, on the opposite side of the active site cleft, serves as the site of the hydrophobic contact in the case of the F131V mutant. 2-Fluorinated compounds bind more tightly, perhaps due to the field effect of the nearby fluorine on the acidity of the amide proton.

Inactivation studies of acetylcholinesterase with phenylmethylsulfonyl fluoride.

Acetylcholinesterase (AChE), a serine hydrolase, is potentially susceptible to inactivation by phenylmethylsulfonyl fluoride (PMSF) and benzenesulfonyl fluoride (BSF). Although BSF inhibits both mouse and Torpedo californica AChE, PMSF does not react measurably with the T. californica enzyme. To understand the residue changes responsible for the change in reactivity, we studied the inactivation of wild-type T. californica and mouse AChE and mutants of both by BSF and PMSF both in the presence and absence of substrate. The enzymes investigated were wild-type mouse AChE, wild-type T. californica AChE, wild-type mouse butyrylcholinesterase, mouse Y330F, Y330A, F288L, and F290I, and the double mutant T. californica F288L/F290V (all mutants given T. californica numbering). Inactivation rate constants for T. californica AChE confirmed previous reports that this enzyme is not inactivated by PMSF. Wild-type mouse AChE and mouse mutants Y330F and Y330A all had similar inactivation rate constants with PMSF, implying that the difference between mouse and T. californica AChE at position 330 is not responsible for their differing PMSF sensitivities. In addition, butyrylcholinesterase and mouse AChE mutants F288L and F290I had increased rate constants ( approximately 14 fold) over those of wild-type mouse AChE, indicating that these residues may be responsible for the increased sensitivity to inactivation by PMSF of butyrylcholinesterase. The double mutant T. californica AChE F288L/F290V had a rate constant nearly identical with the rate constant for the F288L and F290I mouse mutant AChEs, representing an increase of approximately 4000-fold over the T. californica wild-type enzyme. It remains unclear why these two positions have more importance for T. californica AChE than for mouse AChE.

Pacemaker lead infection: report of three cases and review of the literature.

Pacemaker lead infection is a rare condition, most often occurring when intervention is needed after pacemaker implantation. Diagnosis is by blood cultures and confirmation by transoesophageal echocardiography; transthoracic echocardiography is often inadequate. A literature review indicated the microorganism most responsible for late lead infection is Staphylococcus epidermidis (which can grow on plastic material). A retrospective analysis of patient files from the authors' institution (1993-97) yielded three patients with proven pacemaker lead endocarditis. The diagnosis of pacemaker endocarditis was by transoesophageal echocardiography. The endocarditis appeared after a long period and in two of the three patients there was S epidermidis infection. Thoracotomy with removal of the infected system was performed because of the large dimensions of the vegetations. A new pacemaker was implanted: in one patient with endocardial leads, in the other two with epicardial leads. All three patients recovered well and follow up was uneventful for at least one year.

5'-Azido-N-1-Napthylphthalamic Acid, a Photolabile Analog of N-1-Naphthylphthalamic Acid : Synthesis and Binding Properties in Curcurbita pepo L.

A photolabile analog of N-1-naphthylphthalamic acid (NPA), 5'-azido-N-1-naphthylphthalamic acid (Az-NPA), has been synthesized and characterized. This potential photoaffinity label for the plasma membrane NPA binding protein competes with [(3)H]NPA for binding sites on Curcurbita pepo L. (zucchini) hypocotyl cell membranes with K(0.5) = 2.8 x 10(-7) molar. The K(0.5) for NPA under these conditions is 2 x 10(-8) molar, indicating that the affinity of Az-NPA for the membranes is only 14-fold lower than NPA. While the binding of Az-NPA to NPA binding sites is reversible in the dark, exposure of the Az-NPA treated membranes to light results in a 30% loss in [(3)H]NPA binding ability. Pretreatment of the membranes with NPA protects the membranes against photodestruction of [(3)H]NPA binding sites by Az-NPA supporting the conclusion that Az-NPA destroys these sites by specific covalent attachment.

Electrostatic control of enzyme reactions: the mechanism of inhibition of glucose oxidase by putrescine.

The interaction of putrescine dihydrochloride with glucose oxidase is reported. At pH 7.65 glucose oxidase is strongly anionic (Z = -80). The pKa of an essential acidic group on the reduced form of the enzyme is extremely sensitive to ionic strength, as predicted by simple electrostatic theory [J. G. Voet, J. Coe, J. Epstein, V. Matossian, and T. Shipley (1981) Biochemistry 20, 7182-7185]. Putrescine dihydrochloride was found to inhibit glucose oxidase at pH 7.65 at a constant ionic strength of 0.05. The kinetics do not obey simple competitive inhibition, however. The data can best be explained by a model in which change in the electrostatic potential of the enzyme on putrescine binding changes the observed pKa of the essential acidic group. The pH dependence of putrescine inhibition supports this interpretation. At I = 0.05, 5 mM putrescine was found to change the pKa of the essential acidic group from 7.6 to 7.1. The shift in the pKa as a function of putrescine concentration at pH 7.7 and I = 0.05 also supports the model presented. The Ka for putrescine to the active form of the enzyme was calculated to be 4.2 mM.

Fluorescence energy transfer between cobra alpha-toxin molecules bound to the acetylcholine receptor.

An approach was developed with steady state fluorescence energy transfer measurements to examine the spatial relationship between the two alpha-toxins bound to the acetylcholine receptor. By taking advantage of the slow dissociation rates of alpha-toxins (Naja naja siamensis 3) from the receptor and of the equal probability with which alpha-toxins bind to the two alpha-toxin-binding sites, we derived an equation which allows prediction of a "true" efficiency of transfer based on the relationship between fractional site occupancy and the observed transfer efficiency ascertained from donor quenching. Using this approach, we examined the efficiency of energy transfer between two fluorescently labeled alpha-toxins, N epsilon-fluorescein isothiocyanate lysine 23 alpha-toxin and monolabeled tetramethylrhodamine isothiocyanate alpha-toxin bound to the receptor from the Torpedo californica electric organ. Significantly greater (32 versus 14%) energy transfer was observed with the membrane-associated than with the solubilized receptor, suggesting that transfer between fluorophores on separate receptor molecules is greater than that occurring intramolecularly between the two sites on the receptor. The magnitude of the distances calculated from the intrareceptor energy transfer efficiency combined with the considerable inter-receptor energy transfer indicate that the fluorophores would reside on the outer perimeter of the receptor molecule rather than near the central axis perpendicular to the plane of the membrane.

Electrostatic control of enzyme reactions: effect of ionic strength on the pKa of an essential acidic group on glucose oxidase.

The dissociation constant of an essential acidic group on the reduced form of glucose oxidase from Aspergillus niger (K4) has been found to be extremely sensitive to ionic strength. Increasing the ionic strength from 0.025 to 0.225 causes a decrease in pK4,obsd of 0.9 pH unit, from 8.2 to 7.3. Analysis of the ionic strength dependence of pK4,obsd, making the assumption that the enzyme is a homogeneously charged impenetrable sphere [Edsall, J. T., & Wyman, J. (1958) Biophysical Chemistry, Vol. 1, pp 282-289, 512-514, Academic Press, New York], predicts that the intrinsic pKa of the acidic group is 6.7 and that the charge on the protein is -78. The enzyme was titrated from its isoelectric point (pH 4.05) to pH 7.7, the pH at which the ionic strength dependence was determined. It was found to have an actual charge at that pH of -77, in remarkable agreement with the theoretical prediction. Thus, glucose oxidase exerts electrostatic control on pK4,obsd as though it were a uniformly charged sphere. The group responsible for pK4,obsd has not been identified. However, its measured delta H degrees obsd of 8.0 kcal mol-1 and delta S degrees obsd of -6.1 cal mol-1 K-1, together with its pKa of 6.7, are consistent with the group being a histidine residue.

Deduction of kinetic mechanism in multisubstrate enzyme reactions from tritium isotope effects. Application to dopamine beta-hydroxylase.

Primary tritium isotope effects have been measured for the hydroxylation of [2-3H]dopamine catalyzed by dopamine beta-hydroxylase. Experimental values vary from 8.8 +/- 1.4 at 0.02 mM oxygen to 4.1 +/- 0.6 at 1.0 mM oxygen. It is shown that the observed dependence of the isotope effect on oxygen concentration provides unequivocal evidence for a kinetically significant dissociation of both dopamine and oxygen from enzyme . ternary complex. This approach, which is applicable to any multisubstrate enzyme characterized by detectable kinetic isotope effects, provides an alternate to classical methods for the elucidation of kinetic order in enzyme-catalyzed reactions.

Stereochemical evidence for the evolution of pyridoxal-phosphate enzymes of various function from a common ancestor.

Several pyridoxal-phosphate-dependent enzymes can convert the bound cofactor to pyridoxamine phosphate. This conversion may be an obligatory part of the normal catalytic sequence, as with transaminases, or may be an abnormal path, inactivating the enzyme. This conversion requires protonation of the C(4)' carbon of the cofactor, which has now been shown to proceed stereospecifically and with the same absolute stereochemistry in seven quite different pyridoxal-phosphate enzymes. We report on one of these, tryptophan synthase B protein. This regularity in protonation stereochemistry suggests a remarkable regularity in the geometry of cofactor binding to the apoenzyme. This regularity is interpreted as evidence for the evolution of this entire family of enzymes from a common progenitor which, through the course of evolution, could not invert its original, arbitrary binding stereochemistry without passing through catalytically inactive conformations.