Network


Latest external collaboration on country level. Dive into details by clicking on the dots.

Hotspot


Dive into the research topics where Kenneth R. Hanson is active.

Publication


Featured researches published by Kenneth R. Hanson.


Archives of Biochemistry and Biophysics | 1970

l-Phenylalanine ammonia-lyase: IV. Evidence that the prosthetic group contains a dehydroalanyl residue and mechanism of action

Kenneth R. Hanson; Evelyn A. Havir

Abstract l -Phenylalanine ammonia-lyase from potato tubers upon reduction with tritiated NaBH4 gave Type I tritiated enzyme. The nature of the reduction procedure, the observed correlation between loss of enzyme activity and tritium incorporation, and the magnitude of the incorporation suggested that reduction of an electrophilic center at the active site took place, and that one atom of hydrogen from borohydride is incorporated per active site reduced. Upon HCl hydrolysis, Type I tritiated enzyme gave exchangeable tritium and dl alanine-t in which most, but probably not all, of the tritium was in the β-position. It is proposed that the active site contains a dehydroalanyl residue which upon reduction yields a tritiated alanyl residue so substituted at the nitrogen atom that HCl hydrolysis leads to racemization and tritium loss. The unusual chemical environment of the alanyl residue is indicated by the mildness of the hydrolysis conditions necessary to release dl -alanine-β-t and the observation that Type I tritiated enzyme upon dilution and dialysis yielded exchangeable tritium and a new form of tritiated enzyme (Type II) which gave a neutral tritiated substance upon HCl hydrolysis. Guanidine hydrochloride also brings about the Type I to Type II conversion. The enzyme functions catalytically by converting the NH3+ of l -phenylalanine into a better leaving group. If the nitrogen of the dehydroalanyl residue is present as a Schiffs base masked against attack by such reagents as borohydride, then it is probable that the amino group of phenylalanine adds to the β-position of the dehydroalanyl double bond. If this intermediate undergoes a prototropic shift the conjugated system OCCαCβN may be formed (the three carbon atoms are those of the original dehydroalanyl residue and the nitrogen belongs to phenylalanine). With such a leaving group the free energy of the transition state in the elimination process should be lowered by resonance.


Archives of Biochemistry and Biophysics | 1970

l-Phenylalanine ammonia-lyase

Kenneth R. Hanson; Evelyn A. Havir

Abstract l -Phenylalanine ammonia-lyase from potato tubers upon reduction with tritiated NaBH4 gave Type I tritiated enzyme. The nature of the reduction procedure, the observed correlation between loss of enzyme activity and tritium incorporation, and the magnitude of the incorporation suggested that reduction of an electrophilic center at the active site took place, and that one atom of hydrogen from borohydride is incorporated per active site reduced. Upon HCl hydrolysis, Type I tritiated enzyme gave exchangeable tritium and dl alanine-t in which most, but probably not all, of the tritium was in the β-position. It is proposed that the active site contains a dehydroalanyl residue which upon reduction yields a tritiated alanyl residue so substituted at the nitrogen atom that HCl hydrolysis leads to racemization and tritium loss. The unusual chemical environment of the alanyl residue is indicated by the mildness of the hydrolysis conditions necessary to release dl -alanine-β-t and the observation that Type I tritiated enzyme upon dilution and dialysis yielded exchangeable tritium and a new form of tritiated enzyme (Type II) which gave a neutral tritiated substance upon HCl hydrolysis. Guanidine hydrochloride also brings about the Type I to Type II conversion. The enzyme functions catalytically by converting the NH3+ of l -phenylalanine into a better leaving group. If the nitrogen of the dehydroalanyl residue is present as a Schiffs base masked against attack by such reagents as borohydride, then it is probable that the amino group of phenylalanine adds to the β-position of the dehydroalanyl double bond. If this intermediate undergoes a prototropic shift the conjugated system OCCαCβN may be formed (the three carbon atoms are those of the original dehydroalanyl residue and the nitrogen belongs to phenylalanine). With such a leaving group the free energy of the transition state in the elimination process should be lowered by resonance.


Phytochemistry | 1966

Chlorogenic acid biosynthesis : Incorporation of [α-14C]cinnamic acid into the cinnamoyl and hydroxycinnamoyl conjugates of the potato tuber

Kenneth R. Hanson

Abstract Experiments in which potato tuber slices were maintained on a tracer amount of [α- 14 C]cinnamic acid and synthetic 3- 0 -cinnamoylquinic acid (pH adjusted to 6) showed that if this conjugate is a free intermediate in chlorogenic acid (3- 0 -caffeoylquinic acid) formation, then the active pool of the compound is very small and is contained within a cell compartment whose membrane is relatively permeable to phenylalanine and cinnamic acid but not to the conjugate itself. The specific activities of 3- 0 -cinnamoyl-, 3- 0 - p -coumaroyl-, and 3- 0 -caffeoylquinic acid; 3- 0 - p -coumaroyl-, and 3- 0 -caffeoylshikimic acid; and 1- O - p -coumaroyl-, and 1- O -caffeoyl-β- d -glucose were determined in this experiment. The results of similar experiments on which tuber slices were maintained on [α- 14 C]cinnamic acid and quinic acid, or phenylalanine and quinic acid together with a tracer amount of [α- 14 C]cinnamic acid, are reported. The specific activities of the isolated conjugates indicate that in each series the caffeoyl derivatives are formed by the hydroxylation of the p -coumaroyl derivatives. The specific activity data also suggest that cinnamic acid and the derived conjugates are formed within a compartment or compartments of the cell.


The Enzymes | 1972

3 The Enzymic Elimination of Ammonia

Kenneth R. Hanson; Evelyn A. Havir

Publisher Summary This chapter compares the four enzymes catalyzing the eliminations of ammonia, which include aspartate ammonia-lyase, 3-methylaspartate ammonia-lyase, histidine ammonia-lyase, and phenylalanine ammonia-lyase. The first cell-free preparation of aspartate ammonia-lyase was made from Bacillus fluorescens liquifaciens . Its presence has been demonstrated in many bacteria, fungi, a few higher plants, and in the livers of sharks, ducks, chickens, and frogs. The enzyme presumably allows microorganisms to use aspartate as a carbon source, however it may also have a synthetic function. At alkaline pH values the enzymes from Bacterium cadaveris and E. coli show sigmoidal kinetics. 3-Methylaspartate ammonia-lyase shows Michaelis-Menten type of kinetics at all pH values studied, although complications arise as it requires monovalent and divalent metal ions for activity. Evidence for the participation of an –SH group in the catalytic mechanism has been obtained from studies of the protection afforded by substrates and inhibitors against inactivation by sulfhydryl reagents. In mammals, the histidine ammonia-lyase is found in the skin as well as in the liver. The metabolic defect, histidinemia, has been described in children and has been shown to result from an absence of histidine ammonia-lyase activity. In bacteria, the enzyme participates in the catabolism of exogenous histidine. The enzyme has been extensively purified from Pseudomonas species in several laboratories. Phenylalanine ammonia-lyase is the only ammonia-lyase, which is widely distributed in plants. In higher plants, the enzyme diverts phenylalanine and tyrosine from protein synthesis to such phenylpropanoid compounds as lignin, flavonoids, conjugates of the hydroxycinnamic acids, and certain alkaloids. In microorganisms, the enzyme functions in the catabolism of exogenous amino acids, the cinnamic and p-coumaric acids formed being used as a carbon source.


Methods in Enzymology | 1970

[72a] l-phenylalanine ammonia-lyase (potato tubers)☆

Evelyn A. Havir; Kenneth R. Hanson

Publisher Summary This chapter presents the assay, purification, and properties of L-phenylalanine ammonia-lyase from potato tubers. The initial rate of cinnamate formation is followed with a spectrophotometer. Under the standard assay conditions, 1 micromole of trans-cinnamate in 3 ml has an absorbancy of 3.0 at 290 m μ and 6.1 at 270 m μ . The standard assay is performed at the optimum pH with an amount of substrate giving close (96%) to the saturation velocity of the enzyme; all results are corrected to 30°. Protein may be determined by the biuret method in the early stages and spectrophotometrically in the later stages of purification. The bell-shaped curve for initial velocity at saturating substrate concentration as a function of pH may be defined in terms of the apparent dissociation constants of an enzyme–substrate complex. The enzyme catalyzes the formation of cinnamate from phenylalanine but the rate is 1/5000 or less of the rate with L-phenylalanine.


Archives of Biochemistry and Biophysics | 1986

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

Kenneth R. Hanson; Richard B. Peterson

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.


Archives of Biochemistry and Biophysics | 1981

Phenylalanine ammonia-lyase: Mirror-image packing of d- and l-phenylalanine and d- and l-transition state analogs into the active site

Kenneth R. Hanson

Abstract The binding of substrate and product analogs to phenylalanine ammonia-lyase (EC 4.3.1.5) from maize has been studied by a protection method. The ligand dissociation constants, K L , were estimated from the variation with [L] of the pseudo-first-order rate constants for enzyme inactivation by nitromethane. The phenylalanine analogs d - and l -2-aminooxy-3-phenylpropionic acid showed K L , values over 20,000-fold lower than the K m for l -phenylalanine. From these and other K L values it is deduced that when the enzyme binds l -phenylalanine the structural free energy stored in the protein is higher than when it binds the superinhibitors. Models for binding d - and l -phenylalanine and the superinhibitors are described. The enantiomeric pairs are considered to have similar K L values because they pack into the active site in a mirror-image relationship. If the elimination reaction approximates to the least-motion course deduced on stereoelectronic grounds, the mirror-image packing of the superinhibitors into the active site mimics the conformation inferred for a transition state in the elimination. It appears, therefore, that structural changes take place in the enzyme as the transition state conformation is approached causing stored free energy to be released. This lowers the activation free energy for the elimination reaction and accounts for the strong binding by the above analogs.


Archive | 1979

An Introduction to the Enzymology of Phenylpropanoid Biosynthesis

Kenneth R. Hanson; Evelyn A. Havir

It is the first purpose of this review to provide an outline of phenylpropanoid biosynthesis that can serve as an introduction to the work described in the next few chapters. The enzymology of three early steps in the metabolic pathway will be considred in some detail. These short reviews both point to recent findings and exemplify the progress, difficulties, and goals of the field. One purpose for studying the enzymology of such steps in detail is to make it possible for the plant physiologist to investigate the living plant in a rational and productive way. An enormous amount of painstaking and excellent work has already been carried out on the effects of light, hormones, temperature, infection, and development on various aspects of phenylpropanoid metabolism.11 8 99 100No attempt will be made to review this material, which falls within the scope of a later chapter, but questions of relevance to physiological studies will be discussed where they are suggested by the enzymology.


Archives of Biochemistry and Biophysics | 1981

Phenylalanine ammonia-lyase: A model for the cooperativity kinetics induced by d- and l-phenylalanine

Kenneth R. Hanson

Abstract A preparation of l -phenylalanine ammonia-lyase (EC 4.1.3.5.) from soybean ( Glycine max L. cv. Kanrich) showed negative cooperativity with respect to l -phenylalanine and competitive inhibition by d -phenylalanine. A two-protomer partially concerted model for inhibition kinetics is described. If cooperativity is associated with ligand binding but not k cat , plots of v against log [S] at constant [I] are symmetrical. Such curves may be fitted by graphical or iterative least-squares methods. The experimental results conform to this restricted model. The three-substrate and three-inhibitor dissociation constants were estimated by a stepwise procedure. For substrate only the first and second dissociation constants were 12 and 78 μ m , respectively, with a symmetry point value of 30.5 μ m . To a first approximation, site occupancy determines the cooperativity. As d - and l -phenylalanine produce equivalent effects, they are assumed to pack into the same induced space. As ligand binding at one site has little influence on the relative d : l binding at the other and does not influence k cat , cooperativity probably reflects changes in regions remote from the active site such as the interface between the protomers. The regulatory range in [S] of the enzyme in vivo may be indicated by the linearity range of the semilog plot for the isolated enzyme. The observed range corresponds to a 100-fold change in [S] compared to a 10-fold change for Michaelis-Menten kinetics.


Archives of Biochemistry and Biophysics | 1977

L-Phenylalanine ammonia-lyase (maize, potato, and Rhodotorula glutinis) Explaining the kinetic effects of substrate modification by linear free-energy relationships.

Kenneth R. Hanson; Evelyn A. Havir

Abstract The action of phenylalanine ammonia-lyase [EC 4.3.1.5] on a series of para-substituted l -phenylalanines has been investigated. Multiple linear regression analysis has been used to relate the logs of kcat, Km, and k cat K m to substituent parameters for electron withdrawal, hydrophobic bonding, and size. The inhibitory action of the enantiomeric d -phenylalanines was also investigated. The results indicate that the rate-limiting step is not subsequent to the release of cinnamate from the enzyme. Explanations for the observed regression constants are discussed in terms of the influence of the substituent parameters on the dissociation constant for the bound substrate, the rate-limiting step, and intermediate steps such as the elimination process. The discussion utilizes a new theoretical treatment of the application of linear free-energy relationships to steady-state enzyme kinetics. It is shown that, in order to interpret structure-activity correlations in terms of rate and equilibrium constants for an unbranched catalytic sequence, a restricted model must apply. The reaction must have a single rate-limiting step so that quasi-equilibrium conditions prevail. In the QE-DS (quasi-equilibrium dominant-state) model, a single state of the enzyme-substrate complex is assumed to predominate. In the QE-FR (quasi-equilibrium fixed-ratio) model, changes in the enzyme substituents are assumed not to alter the ratio between the different forms of the enzyme-substrate complex prior to the rate-limiting step.

Collaboration


Dive into the Kenneth R. Hanson's collaboration.

Top Co-Authors

Avatar
Top Co-Authors

Avatar

Richard B. Peterson

Connecticut Agricultural Experiment Station

View shared research outputs
Top Co-Authors

Avatar
Top Co-Authors

Avatar

Neil A. McHale

Connecticut Agricultural Experiment Station

View shared research outputs
Top Co-Authors

Avatar

Hans Hirschmann

Case Western Reserve University

View shared research outputs
Top Co-Authors

Avatar
Top Co-Authors

Avatar

Israel Zelitch

Connecticut Agricultural Experiment Station

View shared research outputs
Top Co-Authors

Avatar

Joseph M. Kornfeld

Oklahoma State Department of Health

View shared research outputs
Top Co-Authors

Avatar

Lester Hankin

Connecticut Agricultural Experiment Station

View shared research outputs
Top Co-Authors

Avatar

Michael E. Salvucci

United States Department of Agriculture

View shared research outputs
Researchain Logo
Decentralizing Knowledge