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K R Hanson

Publications and source records attributed to K R Hanson.

At least 19 recordsLinked to original sources
PubMed1987

Photorespiration stoichiometry in leaves estimated by combined physical and stereochemical methods: allowance for isomerase-catalyzed 3H losses in ribulose bisphosphate regeneration.

We showed previously [K.R. Hanson and R.B. Peterson (1986) Arch. Biochem. Biophys. 246, 332-346] that under steady-state photosynthetic conditions the fraction of ribulose bisphosphate oxidized and the fraction of glycolate carbon photorespired (the stoichiometry of photorespiration) may be estimated in leaves by a combination of physical and stereochemical methods. The calculations assumed that when (3R)-D-[3-3H1,3-14C]glyceric acid is supplied to illuminated leaf discs the only loss of 3H from the combined photosynthetic and photorespiratory system is the result of glycolate oxidase action; i.e., the isomerase-catalyzed losses in the regeneration of ribulose bisphosphate are negligible. The present study of tobacco leaf discs under zero-photorespiration conditions (low O2 and high CO2 concentrations), and also of maize leaf discs, shows that some 3H losses occur (between 8 and 13% of the 3H at C-1 of ribulose 5-phosphate). The calculated loss varied moderately with temperature but did not vary when the flux of ribulose bisphosphate formation was altered by changing the irradiance. The calculated loss under zero-photorespiration conditions, therefore, may be used to calculate ribulose bisphosphate and glycolate partitioning under other conditions. Earlier experiments on the influence of O2 and CO2 concentrations of temperature on the partitioning of ribulose bisphosphate and glycolate have been reexamined. The loss corrections decreased all values for the fraction of ribulose bisphosphate oxidized and increased all values for the stoichiometry of photorespiration. Essentially all stoichiometry values were above the theoretical lower limit of 25%. The previous conclusion that the stoichiometry of photorespiration substantially exceeds 25% at higher O2 concentrations and higher temperatures is unchanged. The results with maize leaf discs implied that there is very little oxidation of ribulose 1,5-bisphosphate under normal-air conditions; i.e., photorespiration is indeed suppressed, not merely hidden, by efficient refixation of CO2.

Carbon Dioxide↗
PubMed1986

Regulation of photorespiration in leaves: evidence that the fraction of ribulose bisphosphate oxygenated is conserved and stoichiometry fluctuates.

Under steady-state conditions the combined system of the reductive photosynthetic cycle and the oxidative photorespiratory loop may be defined by two partitioning terms: the fraction of ribulose bisphosphate oxygenated and the fraction of glycolate carbon photorespired (the stoichiometry of photorespiration). A combination of physical and stereochemical methods [K.R. Hanson, and R. B. Peterson, (1985) Arch. Biochem. Biophys. 237,300-310] has been used to estimate these partitionings for tobacco leaf discs. Inverted discs, as compared to normally oriented discs, were found to have greater net photosynthesis; their ratio of photorespiration to net photosynthesis was less, and less of their glycolate carbon was photorespired. An eightfold reduction of irradiance below that of full sunlight for inverted discs in normal air at 32 degrees C reduced both photosynthesis and photorespiration about threefold but had little effect on the partitioning of ribulose bisphosphate and glycolate. Increasing the temperature from 22 to 40 degrees C for inverted discs in normal air and 1000 microE m-2 s-1 irradiance had little effect on net photosynthesis but increased the ratio of photorespiration to net photosynthesis almost threefold; ribulose bisphosphate partitioning was little changed but the fraction of glycolate carbon photorespired more than doubled. If field-grown plants respond to temperature in a similar fashion, genetic intervention to reduce the increase in photorespiration stoichiometry with temperature could increase total daily carbon assimilation and hence improve crop yields.

Carbon Dioxide↗
PubMed1985

The stoichiometry of photorespiration during C3-photosynthesis is not fixed: evidence from combined physical and stereochemical methods.

The stoichiometry of photorespiration, S, is defined as the fraction of glycolate carbon photorespired. It is postulated that under steady-state conditions there are two determinants of the ratio of photorespiration to net photosynthesis: the partitioning of ribulose bisphosphate between oxidation and carboxylation, and the partitioning of glycolate between reactions leading to complete oxidation to CO2 (S = 100%) and those yielding CO2 plus serine (S = 25%). S may be calculated using two independent probes of the system. The physical probe, using an infrared gas analyzer, measured photorespiration and net photosynthesis, and hence their ratio PR/NPS = pn(phys). The metabolic probe employed tracer (3R)-D-[3-3H1,3-14C]glyceric acid to determine r, the fraction of 3H retained in the triose phosphates leaving the chloroplasts. It is deduced from the postulated model that S = pn(phys) . r/(1 - r). Experiments have been performed with illuminated tobacco leaf discs (inverted) under varying concentrations of O2 and CO2. Increasing O2 at constant CO2 increased pn(phys) and decreased r, whereas increasing CO2 at constant O2 had the opposite effect. S more than doubled at 32 degrees C on going from 16 to 40% O2 (340 microliters CO2/liter) and decreased 40% on going from 200 to 700 microliters CO2/liter (21% O2). For discs in normal air S was somewhat greater than 27%. It is suggested that net photosynthesis, and therefore crop yields, could be increased by selecting for crop plants with reduced photorespiration stoichiometry.

Carbon Dioxide↗
PubMed1984

Stereochemical determination of carbon partitioning between photosynthesis and photorespiration in C3 plants: use of (3R)-D-[3-3H1, 3-14C]glyceric acid.

When (3R)-D-[3-3H1,3-14C]glyceric acid is supplied in tracer amounts to illuminated tobacco leaf discs, the acid penetrates to the chloroplasts without loss of 3H, and is phosphorylated there. Subsequent metabolism associated with the reductive photosynthetic cycle fully conserves 3H. Oxidation of ribulose bisphosphate (RuBP) by RuBP carboxylase-oxygenase (EC 4.1.1.39) results in the formation of (2R)-[2-3H1, 14C]glycolic acid which, on oxidation by glycolate oxidase (EC 1.1.3.1), releases 3H to water. Loss of 3H from the combined photosynthetic and photorespiratory systems is, therefore, associated with the oxidative photorespiratory loop. Assuming steady-state conditions and a basic metabolic model, the fraction of RuBP oxidized and the photorespiratory carbon flux relative to gross or net CO2 fixation can be calculated from the fraction of supplied 3H retained in the triose phosphates exported from the chloroplasts. This retention can be determined from the 3H:14C ratio for glucose obtained from isolated sucrose. The dependence of 3H retention upon O2 and CO2 concentrations can be deduced by assuming simple competitive kinetics for RuBP carboxylase-oxygenase. The experimental results confirmed the stereochemical assumptions made. Under conditions of negligible photorespiration 3H retention was essentially complete. The change in 3H retention with O2 and CO2 concentrations were investigated. For leaf discs (upper surface up) in normal air, it was estimated that 39% of the RuBP was oxidized, 32% of the fixed CO2 was photorespired, and the photorespiration rate was 46% of the net photosynthetic CO2 fixation rate. These are minimal estimates, as it is assumed that the only source of photorespired CO2 is glycine decarboxylation.

Carbon↗
PubMed1979

Phenylalanine ammonia-lyase: enzymic conversion of 3-(1,4-cyclohexadienyl)-L-alanine to trans-3-(1,4-cyclohexadienyl)acrylic acid.

The phenylalanine analogue 3-(1,4-cyclohexadienyl)-L-alanine is converted to the hitherto unknown cinnamate analogue trans-3-(1,4-cyclohexadienyl)acrylic acid by L-phenylalanine ammonia-lyase (EC 4.3.1.5) from maize, potato, or Rhodotorula glutinis. The structure assigned to the product is confirmed by its 1H nuclear magnetic resonance spectrum and by the chemical synthesis to be described in a subsequent paper. On comparing the above substrate analogue with L-phenylalanine, the Km was lowered only slightly but kcat was reduced 14--40-fold depending on the source of the enzyme. Because the compounds closely resemble each other in size and hydrophobic properties, this lowering of kcat may be attributed to the electronic effect of replacing the pi electrons of the aromatic system by those of a double bond. Correct alignment at the active site appears to depend upon the space-filling properties of the ring system; open chain analogues that retain the gamma, beta double bond were found to be inhibitors, not substrates.

Acrylates↗
PubMed1975

Reactions at prochiral centers. Interdependence in the estimation of enzyme stereospecificity toward prochiral centers and the configurational purity of labeled substrates.

In many enzymic reactions one or the other hydrogen is released from a prochiral center abCH2. To determine the stereospecificity with respect to such centers under nonreversal conditions a substrate with a chiral center abC1HH is employed (H = 3H or 2H). Assuming the simplest possible model, the R specificity of an enzyme or enzyme system (eR) may be defined in terms of the configurational R purity of the substrate with respect to the labeled center (pR), and the fraction of H released (r) or conserved (l - r) during the reaction by the equation eR = [(l - r) -pR]/[1 - 2pR]. Where r is constant, this is a rectangular hyperbola. The apparent stereospecificity calculated with this equation serves as a characteristic of the experimental system irrespective of the assumed model. The problems arising from direct isotope effects are discussed and the theoretical advantages of using product 3H:14C ratios under low conversion conditions noted. Applications of the equation to the determination of configurational purity at labeled centers, to the study of stereospecificity in enzymes and enzyme systems, and to the study of the biosynthesis of natural products are discussed.

Binding Sites↗
PubMed1975

L-phenylalanine ammonia-lyase (maize, potato, and Rhodotorula glutinis). Studies of the prosthetic group with nitromethane.

Highly purified enzyme (EC 4.1.3.5) from Rhodotorula glutinis was shown by sodium dodecyl sulfate gel electrophoresis to have subunits which if not identical are closely similar in molecular weight. Like the enzyme from maize and potato [Havir, E. A., and Hanson, K. R. (1973), Biochemistry 12, 1583] it is a tetramer of molecular weight similar to 4 times 83,000. Enzyme from all three sources inactivated and labeled at the active site with 14-CH3NO2 gave on HCl hydrolysis 14-CO2, H-14-CO2H, D- and L-[14-C]aspartic acid, and unidentified radioactive products. In addition, the labeled R. glutinis enzyme gave [1,2-14-C2]glycine. The formation of the first three products is compatible with the hypothesis that the electrophilic prosthetic group of the enzyme contains the dehydroalanine imine system greater than C equals to N minus C-alpha(equals to C-beta-H2)COminus and inactivation involves attack on C-beta. The second-order rate constants for CH3NO2 inactivation varied with pH as a simple titration curve. The pKa values calculated from the curves for the three enzymes differed and were lower than the pKa of CH3NO2 by at least 1 pH unit. Apparently the inactivation process is enzyme catalyzed. Both inactivation and addition of the substrate amino group may occur with attack on C-beta.

Ammonia-Lyases↗