Eugene E. Dekker

Seed germination studies. II. Pathways for starch degradation in germinating pea seedlings.

Abstract Amylase activity is present in extracts of the axis tissue of etiolated pea seedlings. Several lines of evidence establish that this starch-hydrolyzing activity is due to the presence of a β-amylase (α-1,4-glucan maltohydrolase, EC 3.2.1.2); the properties of this enzyme are reported. Maltase (EC 3.2.1.20) activity is also present in extracts of the same tissues. Since α-amylase (EC 3.2.1.1) activity in the germinating cotyledon, the following hydrolytic pathway for the degradation of starch to glucose is present in the pea seedling: The importance of this pathway is considered in relation to the alternative route for starch catabolism, namely, the phosphorolytic pathway involving the enzyme phosphorylase (EC 2.4.1.1).

Seed germination studies. I. Purification and properties of an alpha-amylase from the cotyledons of germinating peas.

Abstract A starch-hydrolyzing enzyme present in extracts of the cotyledons of germinating peas has been purified over 3400-fold. Several independent criteria show that this activity is due to an amylase of the α-type (α-1,4-glucan-4-glucanohydrolase, EC 3.2.1.1). The enzyme exhibits maximal activity in the pH range 5.3–5.9; the Michaelis constant ( K m ) and energy of activation ( E a ) for the enzyme-catalyzed reaction are 2·10 −4 g soluble starch per ml and 7600 cal/mole, respectively. Calcium ions protect the amylase against heat inactivation, whereas incubation of the enzyme with 5·10 −4 M EDTA for 20 min at room temperature results in complete loss of activity. With amylose and amylopectin as substrates, this enzyme is similar in action pattern to other known plant α-amylases.

Carboxylation of β-methylcrotonyl coenzyme A by a purified enzyme from chicken liver

In an earlier s tudy 1, 2 of leucine metabolism in crude heart extracts, da ta were obtained indicating that fl-methylcrotonyl CoA is acted upon by enoyl hydrase (crotonase) and tha t the resulting fl-hydroxyisovaleryl CoA is carboxylated enzymically in the presence of ATP to yield fl-hydroxy-fl-methylglutaryl CoA. This product is known 8 to be a t tacked by a specific cleavage enzyme to finnish equimolar amounts of acetoacetate and acetyl CoA. LYNEN AND KNAPPE 4-6 have recently made the interesting discovery, however, tha t fl-methylcrotonyl CoA is carboxylated directly in crotonase-free enzyme preparations from Mycobacterium spp. fl-Methylglutaconyl CoA, the product of this carboxylation reaction, is hydra ted to yield fl-hydkoxy-fl-methylglutaryl CoA by the action of methylglutaconase, a specific hydrase present in microorganisms and in animal tissues 7. During the past two years we have been engaged in the purification of the relatively unstable carboxylase from chicken liver in the hope tha t interfering enzymes could be removed and the nature of this reaction in animal tissues could be established with finality. Wi th a Ioo-fold purified preparation of the carboxylase, a requirement for methylglutaconase in the overall conversion of f l-methylcrotonyl CoA to acetoacetate has been established (Table I). Furthermoie, by employing carboxylase and cleavage-enzyme preparations preincubated with p-chloromercuribenzoate to inhibit the residual crotonase, fl-methylcrotonyl CoA, rather than r: hydroxyisovaleryl CoA, has been identified as the true substrate for the chicken-liver carboxylase (Table II) . The further unexpected finding has been made that fl-methylvinylacetyl CoA also gives rise to acetoacetate in this carboxylase system in the absence of added crotonase. The absorption maximum characteristic of a,fl-unsaturated thiol esters (~t = 267 m~ for f l-methylcmtonyl CoA) does not appear when

A Modified, High Yield Procedure for the Synthesis of Unlabeled and 14C-Labeled 4-Methylene-DL-glutamic Acid

This paper describes the complete chemical synthesis of 4-methylene-DL-glutamic acid from diethylmalonate, formaldehyde and diethyl acetamidomalonate. The amino acid was obtained pure following ion-exchange chromatography and/or crystallization from hot water in an overall yield of 30% based on the amount of diethylmalonate used. Several physico-chemical characteristics of the synthetic compound were determined, including ir and pmr spectra, chromatography on paper, retention time on an amino acid analyzer, pK values and melting point; all properties of the synthetic material were found to be identical to those seen with the naturally occurring L-isomer. The procedure for obtaining gram quantities of the unlabeled compound has also been modified for the synthesis of high specific activity (10.6 mCi/mol) 4-methylene-[2-14C]-DL-glutamic acid.

Functional analysis of E. coli threonine dehydrogenase by means of mutant isolation and characterization

The oxidation of L-threonine to 2-amino-ketobutyrate, as catalyzed by L-threonine dehydrogenase, is the first step in the major pathway for threonine catabolism in both eukaryotes and prokaryotes. Threonine dehydrogenase of E. coli has considerable amino-acid sequence homology with a number of Zn(2+)-containing, medium-chain alcohol dehydrogenases. In order to further explore structure/function interrelationships of E. coli threonine dehydrogenase, 35 alleles of tdh that imparted a no-growth or slow-growth phenotype on appropriate indicator media were isolated after mutagenesis with hydroxylamine. Within this collection, 14 mutants had single amino-acid changes that were divided into 4 groups: (a) amino-acid changes associated with proposed ligands to Zn2+; (b) a substitution of one of several conserved glycine residues; (c) mutations at the substrate or coenzyme binding site; (d) alterations that resulted in a change of charge near the active site. These findings uncover previously unidentified amino-acid residues that are important for threonine dehydrogenase catalysis and also indicate that the three-dimensional structure of tetrameric E. coli threonine dehydrogenase has considerable similarity with the dimeric horse liver alcohol dehydrogenase.

Variant properties of bovine liver 2-keto-4-hydroxyglutarate aldolase; its β-decarboxylase activity, lack of substrate stereospecificity, and structural requirements for binding substrate analogs

1. 1. 2-Keto-4-hydroxyglutarate aldolase (2-oxo-4-hydroxyglutarate glyoxylatelyase; reaction: 2-oxo-4-hydroxyglutarate ⇋ pyruvate + glyoxylate) catalyzes the cleavage and the formation of both optical isomers of 2-keto-4-hydroxyglutarate at the same rate and to the same extent. 2. 2. The specificity of azomethine (Schiff base) formation with this enzyme was studied. Of some forty compounds tested, inactivation in the presence of NaBH4 occurs (in order of decreasing effectiveness) with 2-keto-4-hydroxy-4-methylglutarate, 2-ketoglutarate, 2-keto-4-hydroxybutyrate, 2-keto-3-deoxy-6-phosphogluconate, fructose 1,6-diphosphate, 2-keto-4,5-dihydroxyvalerate, 2-keto-3-deoxygluconate, and 5-keto-4-deoxyglucarate (among 2-keto-4-hydroxyglutarate analogs); only with bromopyruvate and 2-ketobutyrate (among pyruvate analogs); and also with glyoxal, formaldehyde, acetaldehyde, and glycolaldehyde (glyoxylate analogs). In this regard, therefore, a high degree of specificity is shown for pyruvate but not for glyoxylate; this aldolase is also quite specific for analogs of 2-keto-4-hydroxyglutarate having a pyruvate-like structure on one end of the molecule. 3. 3. 2-Keto-4-hydroxyglutarate aldolase actually catalyzes the cleavage of only 2-keto-4,5-dihydroxyvalerate, 2-keto-4-hydroxy-4-methylglutarate, 5-keto-4-deoxyglucarate, 2-keto-3-deoxy-6-phosphogluconate, and 2-keto-4-hydroxybutyrate at 33%, 8%, 3%, 2% and 1%, respectively, the rate of 2-keto-4-hydroxyglutarate cleavage. For certain substrate analogs, therefore, there is a dissociation of azomatheine formation from a concurrent cleavage of that compound. 4. 4. In addition, this purified aldolase was found to catalyze the β-decarboxylation of oxaloacetate at 50% the rate of 2-keto-4-hydroxyglutarate cleavage.

Formation of D-1-amino-2-propanol from L-threonine by enzymes from Escherichia coli K-12.

Abstract Escherichia coli K-12 cells contain two dehydrogenases which in sequence catalyze the net conversion of L -threonine to the D -isomer of 1-amino-2-propanol. These two enzymes are L -threonine dehydrogenase ( L -threonine + NAD + → aminoacetone + CO 2 + NADH + H + ) and D -1-amino-2-propanol dehydrogenase (aminoacetone + NADH + H + D -1-amino-2-propanol + NAD + ). Each enzyme has been obtained in purified form free of the other; the nature of the reaction catalyzed by the latter dehydrogenase alone and in a coupled system with the former enzyme has been studied. The results provide an explanation on the enzymological level for the utilization of L -threonine by cell suspensions of certain microorganisms for the biosynthesis of the D -1-amino-2-propanol moiety of Vitamin B 12 .

Purification of rat-liver γ-hydroxyglutamate transminase and its probable identity with glutamate-aspartate transminase

Abstract 1. 1. An enzyme which catalyzes the transamination of γ-hydroxyglutamate has been purified over 300-fold from rat-liver homogenates. 2. 2. The following observations strongly suggest that γ-hydroxyglutamate transminase is identical with glutamate-aspartate transaminase ( L -aspartate: 2-oxo-glutarate aminotransferase, EC 2.6.1.1): a, at all stages of purification from either rat-liver or pig-heart extracts, the glutamate to γ-hydroxyglutamate transminase ratios are not significantly different; b, transaminase activity for both substrates declines at the same rate during controlled heat denaturation of the purified rat-liver enzyme; c, transamination of γ-hydroxyglutamate is strongly inhibited by either L -glutamate or L -aspartate; d, glutarate and maleate function as competitive inhibitors for either glutamate or γ-hydroxyglutamate and determined K 1 values for a given inhibitor are the same for either amino acid; and e, the purified rat-liver enzyme has the same pH-activity curve for both substrates. 3. 3. The erythro - and threo -isomers of γ-hydroxy- L -glutamate serve as substrates for both isozymes of the rat-liver enzyme as well as for the pig-heart enzyme, although the former isomer is a somewhat better substrate. The corresponding D -diastereo-isomers are enzymically inactive. 4. 4. With γ-hydroxyglutamate, only α-ketoglutarate and oxaloacetate serve as amino group acceptors; pyruvate, α-ketobutyrate, and β-phenylpyruvate are inactive. 5. 5. Other γ-substituted forms of glutamic acid, including γ-methyleneglutamic acid and γ-hydroxy-γ-methylglutamic acid, are also active with the purified enzymes.

Enzymic reactions in mammalian metabolism of γ-hydroxyglutamic acid

Glyoxylic acid has recently been shown to be formed from 7-hydroxyglutamate by an enzyme system present in rat-liver extracts z. Paper-chromatographic identification of alanine as one other product of this process suggested the possibilities of either a direct cleavage of the substrate or a series of reactions with formation of alanine as a terminal product. The present experiments show that the latter alternative is correct. Using glyoxylate formation to measure enzymic activity 1, we have purified the system approx. 4o-fold from dialyzed KCl-ethanol extracts of rat liver. The procedures include acetone precipitation, heat treatment, and the adsorption of inert proteins first by bentonite and then by carboxymethylcellulose. A serious loss (80-90 %)/ of activity is observed when the bentonite-treated fraction is dialyzed exhaustively or passed through columns of Sephadex G-25. After either treatment, full activity is restored by adding heat-deproteinized rat-liver extract. The active component present in this boiled extract has been isolated and shown to be replaceable by and identical with L-glutamine. Other findings substantiate the participation of L-glutamine in the enzymic system. For example, the stimulatory factor present in boiled extract of rat liver is destroyed by glutaminase purified from Escherichia coli extracts. Also, the observed stimulation of glyoxylate formation by added boiled extract of rat liver or L-glutamine is strongly inhibited by the known glutamine antagonists, 6-diazo-5-oxo-norleucine and 7-glutamyl hydrazide. The inhibition shown by either of these two compounds is prevented by the presence of excess L-glut amine. As shown in Table I, L-glutamine greatly accelerates the reaction and only L-isoglutamine and L-glutamate, when present at a level of I ,umole in 3 ml of incubation mixture, partially stimulate glyoxylate formation above the degree of activity

Structural characterization of the zinc site in Escherichia coliL-threonine dehydrogenase using extended X-ray absorption fine structure spectroscopy

Escherichia coliL-threonine dehydrogenase (TDH) is a homotetrameric protein which contains one Zn2+ ion per subunit and is a member of the medium chain, Zn2+-containing alcohol/polyol dehydrogenase family. TDH was subjected to extended X-ray absorption fine structure (EXAFS) spectroscopic analyses to explore what residues might bind the Zn2+; the EXAFS data are consistent with a tetrathiolate ligation sphere for the zinc atom. As a test of this proposed model, the oxidation state of the six cysteine residues in the enzyme was evaluated. Under typical storage conditions (4°C with intermittent exposure to air; 50 mM Tris-HCl buffer, pH 8.4; 5 mM 2-mercaptoethanol, 2-ME) the TDH cysteine residues undergo air-dependent oxidation to form one disulfide bond/subunit with no change in enzymatic activity. Measurements of the free thiol and disulfide levels during storage support this proposal. No disulfide bond forms in TDH when it is stored for up to 100 days at 4°C under argon either with or without 5 mM 2-ME. The previously reported selective reactivity of Cys38 in native TDH towards either iodoacetate or Woodwards reagent K suggests that this residue is not involved in disulfide bond formation. The EXAFS data reported here and the thiol/disulfide levels determined for TDH, together with the homology of TDH to horse liver alcohol dehydrogenase, suggest that the one Zn2+/subunit of TDH is non-catalytic and is probably bound by cysteine residues 93, 96, 99, and 107 in a structural zinc-binding loop of the protein.