W. W. Cleland

Stereoselective preparation of deuterated reduced nicotinamide adenine nucleotides and substrates by enzymatic synthesis

Abstract A-Side (4- R )-(4- 2 H)-reduced nicotinamide adenine dinucleotide (NADD) was prepared by a stepwise oxidation of ethanol- d 6 to acetate in the presence of NAD, alcohol dehydrogenase, and aldehyde dehydrogenase. The B-side (4- S ) isomer of NADD was prepared using the glucose dehydrogenase activity of glucose-6-phosphate dehydrogenase to oxidize to oxidize glucose-1- d in 40% dimethyl aulfoxide. Subsequent purifieation of the reduced nucleotides was achieved using a column of strongly basic polystyrene macroporous resin (AG MP-1) eluted with 0.2 m LiCl, pH 10, and applying the pooled NADD peak to a polyacrylamide gel (Bio-Gel P-2) column. The final A 260 A 340 ratio obtained for these preparations was below 2.3. Preparation of the deuterated reduced nucleotides in this manner allows production of specifieally deuterated substrates by coupled enzymatic synthesis. L -Malate-2- d was prepared by coupled synthesis of A-side NADD to the reduction of oxaloacetate by the A-side enzyme malate dehydrogenase.

Synthesis of various chelating celluloses and their application in removing Al3+ from ATP

Neutron activation analysis of 20 commercial preparations of ATP demonstrated aluminum (a potent inhibitor of hexokinase as Al 3+ -ATP) to be both ubiquitous and the most abundant metal contaminant found. In an effort to find a convenient and effective method of removing Al 3+ from ATP we have synthesized several new chelating celluloses. Several polyvalent car☐ylic acids (EDTA, EGTA, citrate, and aluminon) were linked to aminoethylcellulose via a water-soluble carbodiimide. A chelator similar in chelating ability to ATP, cellulose polyphosphate, was prepared by reaction of excess polyphosphate with cellulose. The ability of these immobilized chelators to compete with ATP for Al 3+ was tested. Cellulose polyphosphate at pH 5 proved to be especially effective in removing Al 3+ from ATP.

Catalytic Mechanism of Perosamine N-Acetyltransferase Revealed by High-Resolution X-ray Crystallographic Studies and Kinetic Analyses

N-Acetylperosamine is an unusual dideoxysugar found in the O-antigens of some Gram-negative bacteria, including the pathogenic Escherichia coli strain O157:H7. The last step in its biosynthesis is catalyzed by PerB, an N-acetyltransferase belonging to the left-handed β-helix superfamily of proteins. Here we describe a combined structural and functional investigation of PerB from Caulobacter crescentus. For this study, three structures were determined to 1.0 Å resolution or better: the enzyme in complex with CoA and GDP-perosamine, the protein with bound CoA and GDP-N-acetylperosamine, and the enzyme containing a tetrahedral transition state mimic bound in the active site. Each subunit of the trimeric enzyme folds into two distinct regions. The N-terminal domain is globular and dominated by a six-stranded mainly parallel β-sheet. It provides most of the interactions between the protein and GDP-perosamine. The C-terminal domain consists of a left-handed β-helix, which has nearly seven turns. This region provides the scaffold for CoA binding. On the basis of these high-resolution structures, site-directed mutant proteins were constructed to test the roles of His 141 and Asp 142 in the catalytic mechanism. Kinetic data and pH-rate profiles are indicative of His 141 serving as a general base. In addition, the backbone amide group of Gly 159 provides an oxyanion hole for stabilization of the tetrahedral transition state. The pH-rate profiles are also consistent with the GDP-linked amino sugar substrate entering the active site in its unprotonated form. Finally, for this investigation, we show that PerB can accept GDP-3-deoxyperosamine as an alternative substrate, thus representing the production of a novel trideoxysugar.

Separation of aldehydes and ketones by chromatography on Dowex-50 in the ethylenediamine form.

Abstract A technique is developed that separates a number of carbonyl-containing compounds on the basis of chemical interaction with ethylenediamine in a column of polystyrene resin (AG-50W-X2) in the ethylenediamine form eluted with 10 m m ethylenediamine-phosphate, pH 8.0, at 25°C. Aldehydes are retained more strongly than ketones on this column. Relative elution volumes for several representative compounds are acetaldehyde > glyceraldehyde > 2,5-anhydromannose (chitose) > cyclohexanone > dihydroxyacetone > glyceraldehyde 3-phosphate > pyruvate > dihydroxyacetone phosphate ≥ glucose, chitose 6-phosphate, and glucose 1-phosphate. Within a given class of compounds the extent of hydration does not appear to affect how well a substance is retained, whereas the presence of negative charge(s) appears to decrease retention. In the absence of anionic functional groups, the retention of carbonyl compounds corresponds roughly to what is known about the relative stability of imines.

Oxamate Is an Alternative Substrate for Pyruvate Carboxylase from Rhizobium etli

Oxamate, an isosteric and isoelectronic inhibitory analogue of pyruvate, enhances the rate of enzymatic decarboxylation of oxaloacetate in the carboxyl transferase domain of pyruvate carboxylase (PC). It is unclear, though, how oxamate exerts a stimulatory effect on the enzymatic reaction. Herein, we report direct (13)C nuclear magnetic resonance (NMR) evidence that oxamate acts as a carboxyl acceptor, forming a carbamylated oxamate product and thereby accelerating the enzymatic decarboxylation reaction. (13)C NMR was used to monitor the PC-catalyzed formation of [4-(13)C]oxaloacetate and subsequent transfer of (13)CO(2) from oxaloacetate to oxamate. In the presence of oxamate, the apparent K(m) for oxaloacetate is artificially suppressed (from 15 to 4-5 μM). Interestingly, the steady-state kinetic analysis of the initial rates determined at varying concentrations of oxaloacetate and fixed concentrations of oxamate revealed initial velocity patterns inconsistent with a simple ping-pong-type mechanism. Rather, the patterns suggest the existence of an alternate decarboxylation pathway in which an unstable intermediate is formed. The steady-state kinetic analysis coupled with the normal (13)(V/K) kinetic isotope effect observed on C-4 of oxaloacetate [(13)(V/K) = 1.0117 ± 0.0005] indicates that the transfer of CO(2) from carboxybiotin to oxamate is the partially rate-limiting step of the enzymatic reaction. The catalytic mechanism proposed for the carboxylation of oxamate is similar to that proposed for the carboxylation of pyruvate, which occurs via the formation of an enol intermediate.

A Kinetic and Isotope Effect Investigation of the Urease-Catalyzed Hydrolysis of Hydroxyurea

The urease-catalyzed hydrolysis of hydroxyurea is known to exhibit biphasic kinetics, showing a rapid burst phase followed by a slow plateau phase. Kinetic isotope effects for both phases of this reaction were measured at pH 6.0 and 25 °C. The observed nitrogen isotope effects for the ammonia leaving group [(15)(V/K)(NH(3))] were 1.0016 ± 0.0005 during the burst phase and 1.0019 ± 0.0007 during the plateau phase, while those for the hydroxylamine leaving group [(15)(V/K)(NH(2)OH)] were 1.0013 ± 0.0005 for the burst phase and 1.0022 ± 0.0003 for the plateau phase. These isotope effects are consistent with a rate-determining step that occurs prior to breaking either of the two possible C-N bonds. The observed carbonyl carbon isotope effects [(13)(V/K)] were 1.0135 ± 0.0003 during the burst phase and 1.0178 ± 0.0003 during the plateau phase. The similarity of the magnitude of the carbon isotope effects argues for formation of a common intermediate during both phases.

A heavy-atom isotope effect and kinetic investigation of the hydrolysis of semicarbazide by urease from jack bean (Canavalia ensiformis).

A kinetic investigation of the hydrolysis of semicarbazide by urease gives a relatively flat log V/ K versus pH plot between pH 5 and 8. A log V m versus pH plot shows a shift of the optimum V m toward lower pH when compared to urea. These results are explained in terms of the binding of the outer N of the NHNH 2 group in semicarbazide to an active site residue with a relatively low p K a ( approximately 6). Heavy-atom isotope effects for both leaving groups have been determined. For the NHNH 2 side, (15) k obs = 1.0045, whereas for the NH 2 side, (15) k obs = 1.0010. This is evidence that the NHNH 2 group leaves prior to the NH 2 group. Using previously published data from the urease-catalyzed hydrolysis of formamide, the commitment factors for semicarbazide and urea hydrolysis are estimated to be 2.7 and 1.2, respectively. The carbonyl-C isotope effect ( (13) k obs) equals 1.0357, which is consistent with the transition state occurring during either formation or breakdown of the tetrahedral intermediate.

13C isotope effect on the reaction catalyzed by prephenate dehydratase.

The (13)C isotope effect for the conversion of prephenate to phenylpyruvate by the enzyme prephenate dehydratase from Methanocaldococcus jannaschii is 1.0334+/-0.0006. The size of this isotope effect suggests that the reaction is concerted. From the X-ray structure of a related enzyme, it appears that the only residue capable of acting as the general acid needed for removal of the hydroxyl group is threonine-172, which is contained in a conserved TRF motif. The more favorable entropy of activation for the enzyme-catalyzed process (25 eu larger than for the acid-catalyzed reaction) has been explained by a preorganized microenvironment that obviates the need for extensive solvent reorganization. This is consistent with forced planarity of the ring and side chain, which would place the leaving carboxyl and hydroxyl out of plane. Such distortion of the substrate may be a major contributor to catalysis.

2-Keto-3-fluoroglutarate: a useful mechanistic probe of 2-keto-glutarate-dependent enzyme systems

2-Keto-3-fluoroglutaric acid prepared by acid hydrolysis of its diethyl ester is stable, as the free acid in aqueous solution at pH 2, and can be stored at -20 degrees C for several years. Both enantiomers are reduced by NADH in the presence of glutamate dehydrogenase (EC 1.4.1.2) to the two diastereomers of 3-fluoro-L-glutamate, which are stable at neutral pH and at high pH unless heated. 2-Keto-3-fluoroglutarate exists in solution almost entirely as a hydrate both at low and neutral pH. Both enantiomers of ketofluoroglutarate react with the pyridoxamine forms of aspartate, alanine and 4-aminobutyrate transaminases to give fluoride release. 2 mol of cosubstrate amino acid react for each mol of ketofluoroglutarate (KFG) when starting from the pyridoxamine form of the enzyme: 2 RCHNH2COOH + KFG + H2O----F- + NH4+ + glutamate + 2 RCOCOOH. Both diastereomers of fluoroglutamate are decarboxylated by glutamate decarboxylase (EC 4.1.1.15) with fluoride release: KFG + H2O----CO2 + F- + HCOCH2CH2COOH. By contrast, only one isomer of fluoroglutamate will react with the pyridoxal form of glutamate-oxalacetate transaminase to give fluoride release: HOOCCHNH2CHFCH2COOH + H2O----4F- + NH4+ + HOOCCOCH2CH2COOH. The enzymatic decarboxylation of 3-fluoroisocitrate produces only one enantiomer of ketofluoroglutarate, which is reduced to threo (2R,3R)-3-fluoroglutamate by NADH and glutamate dehydrogenase: [2R,3S]-HOOCCH(OH)CF(COOH)CH2COOH + NADP+----[3R]-KFG + CO2 + NADPH + H+. The proton, 13C, and 19F-NMR parameters of ketofluoroglutarate and the two fluoroglutamate diastereomers are presented. These molecules are useful probes of enzymatic mechanisms thought to involve carbanion intermediates.

Effect of the presence of a reversible inhibitor on the time course of slow-binding inhibition☆

The half-time for the initial burst seen when a slow-binding inhibitor is present in an enzyme assay decreases from 0.693/k4 to 0.693/(k3 + k4) as the concentration of the slow-binding inhibitor is increased from zero to infinity (k3 and k4 are forward and reverse rate constants for the isomerization causing the slow-binding behavior). If the inhibitor solution contains a classical reversible inhibitor in addition to the slow-binding one, the half-time decreases from the same limit at zero inhibitor to a level which is higher at infinite inhibitor concentration (k3 is divided by (1 + xKi/Kj), where x is the ratio of classical and slow-binding inhibitor concentrations, and Ki and Kj are their initial inhibition constants before the slow-binding phase). Thus if one is using a racemic inhibitor, both enantiomers of which inhibit initially but only one of which shows slow-binding behavior, one will not obtain the correct parameters for the pure slow-binding inhibitor. A similar situation would apply if one were using a mixture of inhibitors such as antibiotics, several of which inhibit initially, but only one of which is a slow-binding inhibitor. This theory is illustrated by determining the half-times for the slow-binding inhibition of yeast hexokinase by various levels of TmATP in the presence and absence of HoATP, which shows little slow-binding behavior.