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D B Northrop

Publications and source records attributed to D B Northrop.

At least 37 records · Page 2Linked to original sources
PubMed1991

Slow step after bond-breaking by porcine pepsin identified using solvent deuterium isotope effects.

The relatively fast artificial substrate Leu-Ser-rho-nitro-Phe-Nle-Ala-Leu-OMe generates a solvent isotope effect of 1.51 +/- 0.02 only on the maximal velocity of peptide hydrolysis catalyzed by porcine pepsin (EC 3.4.23.1). The absence of an isotope effect on V/K places the isotopically-sensitive step after peptide bond cleavage and the release of the first product. Reprotonation of the active site aspartic carboxyls is proposed as the most likely interpretation of this observation. Structural and kinetic similarities between pepsin and other aspartic proteinases, including the therapeutically important targets HIV protease and renin, suggest a similar slow reprotonation step after catalysis. This mechanistic feature has important implications regarding inhibitor design; if most of the enzymes are present in a product-release form during steady-state turnover, then perhaps inhibitors should be designed as product analogs instead of substrate analogs.

Amino Acid Sequence↗
PubMed1989

Quantitative analysis of absorption spectra and application to the characterization of ligand binding curves.

The spectrum of a chromophore may change as a result of perturbations in its environment. The spectral changes resulting from the perturbation are often followed by measurements at just one or two wavelengths but it is usually no more difficult to collect entire spectra. The problem comes in analysing the data from such a series of spectra. In this paper we will suggest a simple procedure in which the spectrum observed under any particular set of conditions may be considered to consist of the sum of two distinct spectral forms. The method, which is free of any assumptions regarding the quantitative relationship between the perturbation and the extent of spectral change, defines any given spectrum in terms of an apparent molar fraction of the contributing spectral forms. The variation of this apparent molar fraction provides information from which a quantitative relationship can be developed to describe the dependence of the spectral change on the perturbant. The method is illustrated using the model system of phenol red protonation and is applied to the characterization of the binding of azide ions to cobalt-substituted carbonic anhydrase.

Azides↗
PubMed1988

Substrate specificities and structure-activity relationships for the nucleotidylation of antibiotics catalyzed by aminoglycoside nucleotidyltransferase 2''-I.

Aminoglycoside nucleotidyltransferase 2''-I (formerly gentamicin adenylyltransferase) conveys antibiotic resistance to Gram-negative bacteria by transfer of AMP to the 2''-hydroxyl group of 4,6-substituted deoxystreptamine-containing aminoglycosides. The kinetics constants of thirteen aminoglycoside antibiotics and the magnesium chelates of eight nucleotide triphosphates were determined with purified enzyme. Eleven of the antibiotics exhibit substrate inhibition attributed to secondary binding of the aminoglycoside to an enzyme-AMP-aminoglycoside complex. Maximal velocities vary by only 4-fold, versus variation of values of Vmax/Km for the aminoglycosides of nearly 4000-fold, consistent with a Theorell-Chance kinetic mechanism as proposed for this enzyme [Gates, C. A., & Northrop, D. B. (1988) Biochemistry (second of three papers in this issue)] with the added specification that the binding of aminoglycosides is in rapid equilibrium. Under these conditions, Vmax/Km becomes kcat/Kd, where kcat is the net rate constant for catalysis (but not turnover) and Kd is the dissociation constant of aminoglycosides from a complex with enzyme and nucleotide. Values of kcat fall closely together into three distinct sets, with the 3',4'-dideoxygentamicins greater than gentamicins greater than kanamycins. These sets reflect unusual structure-activity correlations which are specific for catalysis but have nothing to do with the maximal velocity of this enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Aminoglycosides↗
PubMed1988

Alternative substrate and inhibition kinetics of aminoglycoside nucleotidyltransferase 2''-I in support of a Theorell-Chance kinetic mechanism.

Aminoglycoside nucleotidyltransferase 2''-I conveys multiple antibiotic resistance to Gram-negative bacteria because the enzyme adenylylates a broad range of aminoglycoside antibiotics as substrates [Gates, C. A., & Northrop, D. B. (1988) Biochemistry (preceding paper in this issue)]. The enzyme also catalyzes the transfer of a variety of nucleotides [Van Pelt, J. E., & Northrop, D. B. (1984) Arch. Biochem. Biophys. 230, 250-263]. This doubly broad substrate specificity makes it an excellent candidate for application of the alternative substrate diagnostic [Radika, K., & Northrop, D. B. (1984) Anal. Biochem. 141, 413-417] as a means to determine its kinetic mechanism. The kinetic patterns presented here are composed of one set of intersecting lines and one coincident line and are consistent with a Theorell-Chance kinetic mechanism in which nucleotide binding precedes aminoglycosides, pyrophosphate is released prior to the nucleotidylated aminoglycoside (Q), and turnover is controlled by the rate-limiting release of the final product. Substrate inhibition by tobramycin (B) is partial and uncompetitive versus Mg-ATP, indicating that B binds to the EQ complex, but not in the usual dead-end fashion common to an ordered sequential release of products; instead, Q may escape from the abortive EQB complex at a finite rate. Dead-end inhibition by neomycin C (I) is also partial and uncompetitive versus Mg-ATP but is slope-linear, intercept-hyperbolic, partial noncompetitive versus gentamicin A; both kinetic patterns signify the formation of a partial abortive EQI complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Aminoglycosides↗
PubMed1988

Determination of the rate-limiting segment of aminoglycoside nucleotidyltransferase 2''-I by pH and viscosity-dependent kinetics.

Aminoglycoside nucleotidyltransferase 2''-I follows a Theorell-Chance kinetic mechanism in which turnover is controlled by the rate-limiting release of the final product (Q), a nucleotidylated aminoglycoside [Gates, C. A., & Northrop, D. B. (1988) Biochemistry (second of three papers in this issue)]. The effects of viscosity on the kinetic constants of netilmicin, gentamicin C1, and sisomicin aminoglycoside substrates are as follows: no change in the substrate inhibition constants of all three antibiotics, a small but significant and highly unusual increase in Vmax/Km for netilmicin but large, normal decreases for gentamicin C1 and sisomicin, and marked decreases in the maximal velocities for all three. The lack of effect on substrate inhibition provides essential control experiments, signifying that glycerol does not interfere with binding of aminoglycosides to EQ and that the steady-state distribution of EQ does not increase as the release of Q is slowed by a viscosogen. The decrease in the Vmax/Km of better substrates indicates dominance by a diffusion-controlled component in the catalytic segment, attributed to the release of pyrophosphate. The presence of an increase in the Vmax/Km of the poor substrate, however, is inexplicable in terms of either single or multiple diffusion-controlled steps. Instead, it is here attributed to an equilibrium between conformers of the enzyme-nucleotide complex in which glycerol favors the conformation necessary for binding of aminoglycosides. The decrease in Vmax is consistent with the diffusion-controlled release of the final product determining enzymatic turnover.(ABSTRACT TRUNCATED AT 250 WORDS)

Escherichia coli↗
PubMed1988

Kinetic distinction between rapid-equilibrium random and abortive ordered enzymatic mechanisms using alternative substrates or kinetic isotope effects.

Alternative substrates, such as those isotopically-labeled, which differ in their rate constants of catalysis but not in their rate constants of binding, generate identical values of V/Ka in ordered kinetic mechanisms of bireactant enzymes. This is shown to be true even for the rapid-equilibrium ordered mechanism in which an abortive complex between free enzyme and the second substrate is formed. In contrast, rapid-equilibrium random mechanisms have non-identical values for V/Ka. Consequently, the effect of alternative substrates or isotope effects on V/Ka provides a means to distinguish between these nearly identical kinetic mechanisms.

Enzymes↗
PubMed1987

Preparation and storage of isotopically labeled reduced nicotinamide adenine dinucleotide.

A method for obtaining highly purified NADH in a dry, solid, and stable form is described. The method involves improvements of the ion-exchange and reversed-phase chromatographic procedures of C. J. Newton and S. M. Faynor, and D. B. Northrop (Anal. Biochem., 1983, 132, 50-53). The necessary time to prepare pure NADH has been reduced to a few hours. The final product, obtained by drying the nucleotide from absolute ethanol, shows no detectable decomposition either during the drying procedure or during storage under nitrogen gas at -20 degrees C for several months. Using dry product prepared from fixed volumes of ethanolic solution, standardized solutions of known amounts of the highly purified and stored NADH can be obtained in a few seconds.

Chromatography, High Pressure Liquid↗
PubMed1984

The kinetic mechanism of kanamycin acetyltransferase derived from the use of alternative antibiotics and coenzymes.

Kinetic data for the 6'-aminoglycoside-modifying enzyme, AAC(6')-4, also named kanamycin acetyltransferase, have been collected for six aminoglycoside antibiotics (amikacin, gentamicin C1a, kanamycin A, neomycin B, sisomicin, and tobramycin) and three coenzymes (acetyl-CoA, n-propionyl-CoA, and n-butyryl-CoA). The initial velocity pattern using acetyl-CoA favors a ping-pong kinetic mechanism (Kia = -0.34 +/- 0.34 microM), but the pattern using n-propionyl-CoA supports a sequential one (Kia = 2.7 +/- 0.8 microM). Kinetic analyses using alternative substrates confirm the sequential mechanism and, moreover, clearly identify a random order of addition of antibiotic and coenzyme because V/K values of antibiotics varied 40-fold as the identity of the coenzyme was changed and V/K values of coenzyme varied 13-fold as the identity of the antibiotic was changed. One or both sets of values would have remained unchanged if the mechanism were either ordered sequential or ping-pong. Combining these results with structure-activity data which argue for a rapid rate of release of substrates and products relative to the rate of enzymatic turnover (Radika, K., and Northrop, D. B. (1984) Biochemistry 25, in press) establishes the kinetic mechanism as rapid equilibrium random sequential.

Acetyltransferases↗
PubMed1984

Substrate specificities and structure-activity relationships for acylation of antibiotics catalyzed by kanamycin acetyltransferase.

Antibiotic resistance caused by the presence of the plasmid pMH67 is mediated by the aminoglycoside acetyltransferase AAC(6')-4, also known as kanamycin acetyltransferase. Bacteria harboring the plasmid are resistant to the kanomycins plus a broad range of other deoxystreptamine-containing aminoglycosides but not to the gentamicins XK62-2 and C1 which are substituted at the 6'-position. Substrate specificity studies on the purified enzyme, however, now show that the enzyme acetylates an even broader range of aminoglycosides, including the gentamicins XK62-2 and C1. The enzyme also accepts several acyl-CoA esters, which differ in nucleotide as well as in acyl chain length. Application of the method of analysis of structure-activity data developed earlier for gentamicin acetyltransferase [Williams, J. W., & Northrop, D. B. (1978) J. Biol. Chem. 253, 5908-5914] to the kinetic data obtained for AAC(6')-4 shows that the turnover of the acylation reaction is limited by catalysis and not by the rate of release of either the acetylated antibiotic or CoA. Most structural changes in aminoglycosides cause changes in rates of release, and only drastic changes, near the 6'-amino group, affect catalysis. The structural requirements on aminoglycosides for enzymatic activity run parallel to the structural requirements for antibacterial activity.

Acetyltransferases↗
PubMed1984

Purification of two forms of kanamycin acetyltransferase from Escherichia coli.

Kanamycin acetyltransferase acylates aminoglycoside antibiotics using acetyl-CoA, and thereby conveys bacterial resistance to several clinically important antibiotics, notably amikacin. The enzyme was quantitatively and reproducibly released from Escherichia coli W677 harboring plasmid pMH67 by a modified osmotic shock procedure (bacterial cells are incubated overnight in sucrose and again without sucrose before onset of osmotic shock). The enzyme was purified by dye-ligand chromatography on Affi-Gel Blue in addition to antibiotic affinity chromatography on neomycin-Sepharose-4B. The activity did not increase with subsequent chromatography on ion-exchange, hydrophobic, or molecular-exclusion gels. However, both dye-ligand and molecular-exclusion chromatography, as well as disc-gel electrophoresis, separated the purified enzyme equally into two active protein fractions. Based on the more active of the two forms, the purification was 112-fold with a specific activity of 1.9 IU/mg. The less-active form has an unusual absorbance spectrum, with a maximum near 255 nm, which cannot be explained by the amino acid composition. Chromatography of this form alone regenerated both forms, suggesting that the enzyme is noncovalently conjugated to an uncharged chromophore, such as a lipid. The purified enzyme has a very sharp pH optimum at 5.5 with a plateau on the alkaline side, but is most stable between pH 8.5 and 9.5. Data from electrophoresis in the presence of sodium dodecyl sulfate and gel-filtration on Ultrogel AcA 44 are consistent with a tetrameric protein of 60-70,000 Da.

Acetyltransferases↗
PubMed1984

Purification and properties of gentamicin nucleotidyltransferase from Escherichia coli: nucleotide specificity, pH optimum, and the separation of two electrophoretic variants.

Gentamicin nucleotidyltransferase, AAD 2", catalyzes the transfer of a nucleotide to many aminoglycoside antibiotics, which are the drugs of choice in the treatment of gram-negative bacterial infections. The transfer is accompanied by the production of pyrophosphate, which is coupled to three other enzymes so that an increase in absorbance at 340 nm of NADPH can be monitored continuously as a quantitative assay of activity. A purification method was developed for this enzyme using all common principles of protein purification. These include selection of a desirable source of enzyme (choice of plasmid pMY 10), maximizing cellular yield of enzyme (controlled and monitored growth of Escherichia coli pMY 10/W677), selective extraction of protein (modified osmotic shock), removal of nucleic acids (precipitation with streptomycin sulfate), concentration of protein (precipitation with ammonium sulfate), removal of low-molecular-weight impurities (chromatography on Bio-Gel P-2), separation of proteins on the basis of charge (ion-exchange chromatography on DEAE-Bio-Gel A), separation of proteins according to a biospecific property (affinity chromatography on gentamicin-Affi-Gel), and separation of proteins according to size (gel filtration on Ultrogel AcA 54). Purification to near-homogeneity revealed the presence of two related forms of enzyme. The first had a specific activity of 0.134 units/mg, bound rapidly and tightly to gentamicin-Affi-Gel, eluted as a function of ionic strength from Ultrogel, and migrated faster during electrophoresis in both the presence and absence of sodium dodecyl sulfate. It has an isoelectric point of 5.7 +/- 0.2 and consists of a single polypeptide of 32,500 Da. Kinetic characterization showed a pH optimum of 9.5 and Michaelis constants of 2.76 +/- 0.35 microM for tobramycin, 404 +/- 28 microM for Mg-ATP, 2008 +/- 260 microM for Mg-CTP, 30 +/- 3 microM for Mg-dATP and Mg-dGTP, and 90 +/- 7 microM for Mg-dCTP and Mg-dTTP. The second form had a specific activity of 0.274 unit/mg. It also bound tightly to gentamicin-Affi-gel but the onset of binding was time dependent. This form migrated slower during polyacrylamide gel electrophoresis in both the presence and absence of sodium dodecyl sulfate. It has an isoelectric point of 6.0 +/- 0.2 and consists of a single polypeptide of 31,500 Da. The exact relationship between the two forms has not been elucidated. It is probable that they have a recent common ancestor or are the same polypeptide because the amino acid compositions and polypeptide chain lengths are essentially identical.(ABSTRACT TRUNCATED AT 400 WORDS)

Chemical Phenomena↗
PubMed1984

Correlation of antibiotic resistance with Vmax/Km ratio of enzymatic modification of aminoglycosides by kanamycin acetyltransferase.

Kinetic data for the antibiotic-modifying enzyme kanamycin acetyltransferase AAC(6')-IV have been determined for five aminoglycoside antibiotics (amikacin, gentamicin C1a, kanamycin A, sisomicin, and tobramycin) and compared with close-interval determinations of the minimal inhibitory concentrations of the same antibiotics against Escherichia coli W677 harboring the resistance plasmid pMH67. These minimal inhibitory concentrations for the resistant bacteria varied from 80 to 800 micrograms/ml. Of the kinetic parameters Vmax, Km, and Vmax/Km ratio only Vmax/Km ratio had a linear correlation with minimal inhibitory concentrations (r = +0.818) at pH 7.8, where all antibiotics produced substrate inhibition, but not at pH 6.0, where they did not. The correlation with only Vmax/Km ratio has implications regarding the expression of resistance within the dynamics of the bacterial cell (i.e., antibiotic uptake versus modification), whereas substrate inhibition presents an opportunity to search for new chemotherapeutic agents which will combat resistance directly.

Acetyltransferases↗
PubMed1983

Fitting enzyme-kinetic data to V/K.

Kinetic data from enzyme-catalyzed reactions have been analyzed traditionally in terms of the Michaelis-Menten equation, which assumes that the maximal velocity (V) and the Michaelis constant (K) are the primary kinetic constants. But what is needed from most kinetic studies today is V/K. A new form of the equation is proposed which assumes that V and V/K are the primary kinetic constants: v = (V . S . V/K)/(V + S . V/K). Computer fittings of both experimental and simulated velocity data to both equations give results favoring the new equation.

Computers↗