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P C Kline

Publications and source records attributed to P C Kline.

9 recordsLinked to original sources
PubMed2000

Purification and partial characterization of haloperoxidase from fresh water algae Cladophora glomerata.

Many haloperoxidases have been purified from diverse organisms, including lichen, fungi, bacteria, and marine algae. In this study a haloperoxidase was purified from the fresh water algae, Cladophora glomerata, by homogenization and centrifugation, ammonium sulfate fractionation, ion-exchange and gel filtration chromatography. Molecular weight was determined by SDS-PAGE and by size exclusion HPLC and found to be approximately 43 kDa. The isoelectric point was determined to be approximately 8.1 by isoelectric focusing. The UV spectrum of the peroxidase showed a strong absorbance in the Soret band indicating a heme protein, unlike vanadium-dependent haloperoxidases from marine algae. Fresh water algal haloperoxidase catalyzed the iodination of tyrosine at a pH of 3.1. This haloperoxidase also catalyzes the oxidation of guaiacol and oxidation of iodide as well as catalyzing a peroxide-dependent reaction in both the presence and absence of chloride and bromide ions.

Chlorophyta↗
PubMed1999

Determination of kinetic isotope effects for nucleoside hydrolases using gas chromatography/mass spectrometry.

Kinetic isotope effects are widely used to determine the transition state of chemical and enzymatic reactions. Radioactive isotopes are used most often to determine these kinetic isotope effects. However, stable isotopes offer a number of advantages over the use of radioactive isotopes. These advantages include ease of handling and disposal along with increased safety in the laboratory. [1'-(13)C]Inosine and [1'-(2)H]inosine kinetic isotope effects were determined using a gas chromatograph in conjunction with a mass selective detector for nucleoside hydrolase, a purine-metabolizing enzyme. Three ion pairs were used to determine kinetic isotope effects. These ion pairs were 158/159, 187/188, and 217/218. The average isotope effects for all ion pairs were 1.021 +/- 0.006 for [1'-(13)C]inosine and 1.113 +/- 0.008 for [1'-(2)H]inosine. The transition state consistent with these isotope effects is also consistent with the transition state proposed by Schramm and Horenstein using radioactive substrates.

Animals↗
PubMed1995

Pre-steady-state transition-state analysis of the hydrolytic reaction catalyzed by purine nucleoside phosphorylase.

The slow hydrolytic reaction catalyzed by calf spleen purine nucleoside phosphorylase [Kline, P. C., & Schramm, V. L. (1992) Biochemistry 31, 5964-5973] has been investigated using pre-steady-state kinetic isotope effects and solvolysis studies. The stoichiometric reaction between enzyme and inosine forms 1 mol of free ribose per trimer of purine nucleoside phosphorylase and a tightly bound complex of enzyme and hypoxanthine. The experimental kinetic isotope effects from [1'-3H]-, [2'-3H]-, [4'-3H]-, [5'-3H]-, [1'-14C]-, and [9-15N]inosine are 1.151 +/- 0.004, 1.145 +/- 0.003, 1.006 +/- 0.004, 1.028 +/- 0.005, 1.045 +/- 0.005, and 1.000 +/- 0.005, respectively, for the pre-steady-state conditions. Substrate trapping experiments demonstrated that there is no detectable forward commitment to catalysis for inosine hydrolysis. In contrast, bound inosine is 2.1 times more likely to form product than to dissociate when the enzyme-inosine complex is exposed to saturating PO4. The lack of an observed 9-15N isotope effect is consistent with an internal equilibrium between enzyme-inosine and the enzyme-hypoxanthine-ribose complex in which N9 of hypoxanthine is protonated. The equilibrium occurs as a consequence of slow product release and tightly bound hypoxanthine (Kd = 1.3 x 10(-12) M). This internal equilibrium has a minimal effect on the intrinsic kinetic isotope effects from ribose since equilibrium isotope effects for conversion of inosine to ribose are near unity. When the single-turnover hydrolytic reaction was accomplished in 20% methanol, approximately 85% of the product sugar was 1-methylribose. Under these conditions, the anion-binding pocket fills with solvent which competes for the oxocarbenium ion of inosine formed at the transition state. In the presence of arsenate, no methanolysis of inosine occurs [Kline, P. C., & Schramm, V. L. (1993) Biochemistry 32, 13212-13219]. The results define a transition state with oxocarbenium ion character and weak participation of the attacking solvent nucleophile. Electrostatic potential surfaces of the transition states indicate that arsenate anion is more effective in neutralizing the oxocarbenium ion than is H2O.

Adenosine↗
PubMed1994

Electrostatic potential surfaces of the transition state for AMP deaminase and for (R)-coformycin, a transition state inhibitor.

The transition state for the hydrolysis of AMP by AMP deaminase has been characterized by heavy atom kinetic isotope effects (Merkler, D.J., Kline, P.C., Weiss, P., and Schramm, V.L. (1993) Biochemistry 32, 12993-13001). The experimentally established transition state includes a bond order of 0.8 to the attacking water nucleophile, a full bond order to the exocyclic 6-amino group, rehybridization of C-6 of the purine ring to sp3 and protonation of N-1 by Glu633. The transition state is one the path to formation of an unstable tetrahedral intermediate in which the exocyclic amine undergoes rapid protonation followed by its departure. In this mechanism, the highest energetic barrier on the reaction coordinate is the attack of the zinc-activated water. In a further test of this transition state structure, the electrostatic potential surface for the purine ring of the transition state has been determined by molecular orbital calculations and compared to that of the base of (R)-coformycin 5'-monophosphate, a slow onset, tight binding inhibitor of AMP deaminase that binds with an overall dissociation constant of 10(-11) M. The electrostatic potential surfaces of the aglycones of the transition state and (R)-coformycin are compared to the adenine ring of the substrate and to an alternative transition state structure in which the transition state is late, with fully bonded hydroxyl and fully protonated exocyclic amine. The results indicate a near-match of the electrostatic potential surfaces for the early transition state and (R)-coformycin. The electrostatic nature of the late transition state with a protonated amine leaving group differs both from the transition state determine by kinetic isotope effects and from that of (R)-coformycin analogues. The results provide evidence that the nature of the enzyme-stabilized transition state for adenine deamination involves an early transition state with a partially bonded hydroxyl group. The observed tight binding inhibition by (R)-coformycin analogues as transition state inhibitors results from the similarity of the partial charges on the inhibitors to that of the enzymatic transition state stabilized by AMP deaminase.

AMP Deaminase↗
PubMed1993

Transition-state analysis of AMP deaminase.

The transition state of the allosteric AMP deaminase from Saccharomyces cerevisiae has been characterized by 14C and 15N Vmax/Km heavy-atom kinetic isotope effects. The primary 6-14C isotope effect was measured with [6-14C]AMP, and the 6-15N primary isotope effect was measured by isotope ratio mass spectrometry using the natural abundance of 15N in AMP and by using 15N release from ATP as a slow substrate. Isotope effects for AMP as the substrate were measured in the presence and absence of ATP as an allosteric activator and GTP as an allosteric inhibitor. Kinetic isotope effects with [6-14C]AMP were 1.030 +/- 0.003, 1.038 +/- 0.004, and 1.042 +/- 0.003 in the absence of effectors and in the presence of ATP and GTP, respectively. Isotope effects for [6-15N]AMP averaged 1.010 +/- 0.002. Allosteric activation increased the 15N isotope effect to 1.016 +/- 0.003. A primary 15N kinetic isotope effect with ATP, which has a Vmax/Km 10(-6) that for AMP, was 1.013 +/- 0.001. The presence of D2O as solvent caused a marginally significant decrease in the [6-15N]AMP kinetic isotope effect from 1.011 +/- 0.001 to 1.007 +/- 0.002. Previous studies have established that the solvent D2O effect is inverse (0.34) for slow substrates with two or more protons transferred prior to transition state formation and remains inverse (0.79) with AMP as substrate [Merkler, D. J., & Schramm, V. L. (1993) Biochemistry 32, 5792-5799]. Bond vibrational analysis was used to identify transition states for AMP deaminase that are consistent with all kinetic isotope effects.(ABSTRACT TRUNCATED AT 250 WORDS)

AMP Deaminase↗
PubMed1993

Purine nucleoside phosphorylase. Catalytic mechanism and transition-state analysis of the arsenolysis reaction.

Purine nucleoside phosphorylase from calf spleen catalyzes the arsenolysis of inosine to form hypoxanthine and ribose 1-arsenate, which spontaneously hydrolyzes to ribose and arsenate. In the presence of H2(18)O, no 18O is incorporated into ribose, demonstrating that ribose 1-arsenate hydrolysis occurs by attack of water on the arsenic atom. Rapid reaction kinetics at 20 degrees C result in a biphasic rate curve with the first turnover occurring at a rate of 20 s-1 followed by a steady-state rate of 2 s-1. The product burst is consistent with rapid steps for substrate binding and arsenolysis followed by rate-limiting hypoxanthine release at a rate of 2 s-1. Purine nucleoside phosphorylase with bound [14C]inosine was mixed with excess unlabeled inosine and arsenate to determine relative rates for reaction or dissociation of bound inosine. The commitment factor (product formed/inosine released) was 0.19 at saturating arsenate, indicating that inosine binds to free enzyme and that bound inosine is not in thermodynamic equilibrium with free substrate. At neutral pH, kinetic isotope effects for the phosphorolysis reaction are small, indicating kinetic suppression. Kinetic isotope effects for arsenolysis were measured with [1'-3H]-, [2'-3H]-, [1'-14C]-, [9-15N]-, [4'-3H]-, and [5'-3H]inosine to provide experimental values of 1.118 +/- 0.003, 1.128 +/- 0.003, 1.022 +/- 0.005, 1.009 +/- 0.004, 1.007 +/- 0.003 and 1.028 +/- 0.004 respectively. Following correction for commitment factors, the intrinsic isotope effects were matched to a geometric transition-state model selected by bond-energy bond order vibrational analysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Purine nucleoside phosphorylase. Inosine hydrolysis, tight binding of the hypoxanthine intermediate, and third-the-sites reactivity.

Purine nucleoside phosphorylase from calf spleen is a trimer which catalyzes the hydrolysis of inosine to hypoxanthine and ribose in the absence of inorganic phosphate. The reaction occurs with a turnover number of 1.3 x 10(-4) s-1 per catalytic site. Hydrolysis of enzyme-bound inosine occurs at a rate of 2.0 x 10(-3) s-1 to form a stable enzyme-hypoxanthine complex and free ribose. The enzyme hydrolyzes guanosine; however, a tightly-bound guanine complex could not be isolated. The complex with hypoxanthine is stable to gel filtration but can be dissociated by acid, base, or mild denaturing agents. Following gel filtration, the E.hypoxanthine complex dissociates at a rate of 1.9 x 10(-6) s-1 at 4 degrees C and 1.3 x 10(-4) s-1 at 30 degrees C. The dissociation constant for the tightly-bound complex of enzyme-hypoxanthine is estimated to be 1.3 x 10(-12) M at 30 degrees C on the basis of the dissociation rate. The stoichiometry of the reaction is 1 mol of hypoxanthine bound per trimer. The reaction is reversible since the same complex can be formed from enzyme and hypoxanthine. Addition of ribose 1-phosphate to the complex results in the formation of inosine without release of hypoxanthine. Thus, the complex is catalytically competent. Inorganic phosphate or arsenate prevents formation of the tightly-bound E.hypoxanthine complex from inosine or hypoxanthine. Direct binding studies with hypoxanthine in the presence of phosphate result in 3 mol of hypoxanthine bound per trimer with a dissociation constant of 1.6 microM. In the absence of phosphate, three hypoxanthines are bound, but higher hypoxanthine concentrations cause the release of two of the hypoxanthines with an apparent inhibition constant of 130 microM. The results establish that enzymatic contacts with the nucleoside alone are sufficient to destabilize the N-glycosidic bond. In the absence of phosphate, water attacks slowly, causing net hydrolysis. The hydrolytic reaction leaves hypoxanthine stranded at the catalytic site, tightly bound to the enzyme with a conformation related to the transition state. In the phosphorolysis reaction, ribose 1-phosphate causes relaxation of this conformation and rapid release of hypoxanthine.

Animals↗
PubMedDate not supplied

Simplified guide for precise implant placement: a technical note.

Ideal implant placement is ultimately determined by the requirements of the final restoration. For the surgeon placing implants, proper angulation, parallelism, and spacing are critical to the final restoration. The use of a stainless-steel drill guide sleeve in an acrylic resin surgical template can make it possible to achieve optimal placement. Communication between the restoring dentist, surgeon, and laboratory technician is enhanced by this system since dowel pins fit precisely into the sleeves and determine sleeve and, ultimately, implant position. This system allows the surgeon to negotiate bony irregularities and ramping defects with ease while preparing a more concentric implant site.

Dental Equipment↗