- Email: [email protected]
The measurement of enzyme catalysed rates of reaction by 2D NMR spectroscopy
trends in analytical chemistry,
148
vol. 3, no. 6, 1984
The measurement of enzyme catalysed rates of reaction by 2D NMR spectroscopy James A. Ferretti and Robert S. Balaban Bethesda, MD, USA
Two-dimensional NMR spectroscopy is useful for studying exchange rates of complex enzyme catalysed reactions in the steady state. The technique provides various technical and interpretive advantages over conventional methods and will be usefulfor studying in vivo processes.
The study of exchange rates of enzyme catalysed reactions is an integral part of the characterization of the enzyme. Such studies also contribute to the understanding of the mechanism and control of metabolic processes which are dictated by the flux of high energy phosphates through enzyme catalysed reactions. These reactions rates have historically been studied by stopped flow’, temperature jump2 and radioactive tracer analysis 3. Both stopped flow and temperature jump analyses require that the system initially be placed in a nonequilibrium state, and its return to equilibrium is monitored to obtain the flux or the rate constants. It is often desirable to study these reactions under equilibrium conditions, where data interpretation is sometimes simpler-s, and where studies in vivo can be carried out. Radioactive tracer analysis, which allows a reaction to be studied at equilibrium, requires the introduction of a radiolabel, followed by an appropriate product assay. Recent advances in nuclear magnetic resonance (NMR) spectroscopy and, in particular, the development of two-dimensional Fourier transform NMR (2D-FTNMR) suggest a marked increase in its use. Our purpose is to review the current use of 2DFTNMR to study enzyme catalysed reactions and to indicate potential future developments in the field. NMR is useful for studying reacting systems in dyn
Preparation
0
T X
namic equilibrium. Other advantages of NMR lie in its capability of following an individual nuclear species and in being a non-invasive technique. This latter feature makes NMR unique attractive for in vivo investigations. Enzyme catalysed rates of exchange have been studied by several NMR techniques. One of the first studies was reported by Gadian et ~1.4, who applied line shapes to the study of the isomerization of glucose-6-phosphate. This technique is applicable to simple systems when the exchange rate is of the same magnitude as the chemical shift difference between the exchanging species. Saturation and inversion transfer of magnetization5,6 have been the methods of choice for studying moderately slow enzymatic reactions, since one can investigate reactions with complex pathways. However, these methods require the application of a second irradiating radio frequency (rf) field which can present technical difficulties on some commercial spectrometers and which can limit selectivity in a crowded spectrum. Also, they rely on detection of small differences in peak areas, which limits precision’. One of the recently introduced 2D-techniques is designed for rate studies. This technique was first suggested by Jeener et al.8 and has been applied to the study of enzyme catalysed reaction@. The experiment, which can be performed on most modern NMR spectrometers with no instrumental modifications, consists of three 90 degree pulses as outlined in Fig. 1. The first pulse, followed by an evolution period t,, is used to frequency label the magnetization and generate a second time variable for the 2D Fourier transformation. This period will represent the spin system before exchange takes place. The second or mixing period involves a second 90 degree pulse, an interval rtiX, and a third 90 degree pulse. Any exchange which takes place during Z,ix is easily
n
Evolution
w
0
T X
Detection
Mixing
tzl
I3
Field Gradient Fig. 1. Basic three-pulse sequence for the 20 exchange rate experiment. 01659936/84/$02.00.
@ Elsevier Science Publishers B .V.
trends in analyticalchemistry, vol. 3, no. 6, 1~4
149
represented after 2D-Fourier transformation. The third period, t2, also called the detection period, is used to record the free induction decay (FID). The advantage of this experiment is that the FID contains information on both the initial and final states of the spin system (Fig. 2). The final state is represented by the precession frequencies after the third 90 degree pulse. To complete the representation of the initial state of the system, the interval t, is systematically increased. Fourier transformation (IT) with respect to both t, and t2:
produces a 2D-spectrum which is a function of the two frequency variables F, and F2. Any species which failed to exchange during Z,ix will have the same frequencies along F, and F2. Conversely, species which exchanged during r,ix will have substrate frequencies along F, and product frequencies along F2. These peaks will appear symmetrically at the intersections of the perpendiculars along Fl and F2 and are termed cross peaks. An example of such a situation is shown in Fig. 3 for the anomerization of the a and ,8 forms of glucose-6-phosphate. The cross peaks represent the degree of anomerization which has taken place during t,ix (2.0 s) and they are equal in volume. The anomerization of glucose-6-phosphate, which occurs in the absence of enzyme, is a first order exchange process: cr-glucose-6-phosphate
ep-glucose-6-phosphate
A unique advantage of the 2D-method
is that under
Fig. 2. Schematic representation of the free induction decay for the three pulse experiment on a two site exchange system.
EVOLUTION PERIOD
t-TM’x+ MIXING PERIOD
DETECTION PERIOD
Fig. 3. Stacp plot showing the results of the anomerization of glucose-6-phosphate in the absence of enzyme. The x,,,~~value was 2.0s.
slow exchange conditions the cross peaks represent exclusively species which have undergone exchange. Thus, in a complex spin system all exchange pathways may be observed simultaneously with a single instrument setting. Use of 2D-FTNMR can result in a substantial saving in time or a significant improvement in the precision of the estimate of the rate parameters. Determination of the rate constants requires estimation of peak volumes at various t,ix values. If cross sectional slices through the peaks are Lorentzian, then the volumes may be estimated directly. Alternatively, the volumes may be estimated by taking horizontal areas at various heights above the baselinelo. Rate constants can be obtained from the experimental data using various procedures. In the simpler situations, such as the glucose-6-phosphate anomerization, where the rate equations can be solved analytically, least squares analysis can be employed. The data, together with the results of curve fitting, are shown in Fig. 4. The rate constants obtained by 2D-FTNMR were in excellent agreement with those obtained by classical optical rotation techniques”. For more complex reacting systems, analyses require the assumption of a reaction mechanism which can be difficult to test. An alternative method of data analysis which is quite simple and is useful for complex systems, such as enzyme catalysed reactions, uses measurement of the initial rate of product formation. Use of this initial rate approximation is quite common in the analysis of stopped flow data’*. The cross peak volumes are measured for short T,ix values. This alternative is particularly attractive be-
in analytical
trends
150
7
MIX
=
1.00
chemistry,
vol. 3, no. 6, 1984
20 ,
SEC.
ti 15
10
5
J-----H,;, 0
F*
2
6
4
Fig. 5. A 20 contour plot showing the anomerization and isomerization of glucose-&phosphate and fructose-&phosphate. The tmixvalue was 1.0 s.
n
; I
Fig. 4. Plot of the volumes of the cross peaks (0) and diagonal peaks (0) as a function oft,,, for the glucose-6-phosphate anomerization.
cause of its simplicity and because the cross peaks represent not only the product but also the origin (substrate) associated with a given product. This method was applied to the study of various enzyme catalysed reactions; two examples are the yeast phosphoglucose isomerase catalysed anomerization and the isomerization of glucose-6-phosphate and fructose-6-phosphate, and the adenylate kinase exchange where adenosine diphosphate goes reversibly to adenosine triphosphate and adenosine monophosphate. A 2D-contour plot for the yeast phosphoglucose isomerase reaction is shown in Fig. 5. Cross peaks are apparent for both the anomerization and isomerization reactions. Separate peaks are not observed for the a and p anomers of fructose-6-phosphate because the exchange rates are large compared with chemical shift differences between the resonances of the individual anomers. Systematic variation of Z,ix yields spectra shown in Fig. 6, where one dimensional slices were taken along F2 at the resonance frequency of the fructose-6-phosphate in the F, dimension (see dotted line in Fig. 5). It is in-
teresting to note that isomerization of fructose-6phosphate to a-glucose-6-phosphate begins immediately, whereas isomerization to /?-glucose-6-phosphate occurs only after an induction period. To determine rate constants in the initial rate approximation, we write: =
Xi
kij
E
tmix --) O
where Zij is the normalized cross peak volume, Xi is the mole fraction of substrate, kij is the rate constant, and E is the concentration of free enzyme in solution. The normalized exchange rates, kijE, are presented in Table I. An additional advantage of the 2D-method using the initial rate approximation is that rates are obtained without any knowledge of the spin lattice relaxation times, T,. With some simple assumptions about the reaction mechanisms, one may provide simple interpretations of rate data from 2D-FTNMR which are not readily available from other experimental techniques. For example, if we write the mechanism of the yeast phosphoglucose isomerase reaction as a+E$aE
=PE r\b 7/ FE
=P+E
lb
F+E where a, p, and F refer to the anomers of glucose-6phosphate and fructose-6-phosphate, respectively, we can identify the rate limiting steps in the reaction. Product initially grows linearly for both the anomerization and isomerization reactions, thereby indicating a single slow step for each process. Since the
trends in analyticalchemistry, vol. 3, no. 6, 1984
151
TABLE I. Rates of reaction catalysed by phosphoglucose
isomerase Rate (set-1) (25 “C)
Reaction
a-Glucose-6-phosphate + P-Glucose-6-phosphate /SGlucose-6-phosphate + a-Glucose-6-phosphate Fructose-6-phosphate + a-Glucose-6-phosphate a-Glucose-6-phosphate + Fructose-6-phosphate Fructose-6-phosphate + /3-Glucose-6-phosphate /3-Glucose-6-phosphate -+ Fructose-6-phosphate
anomerization and isomerization occur at widely differing rates (see Table I), only the steps which involve interconversion among the enzyme-substrate complex and the enzyme-product complex (i.e. a E Sj3E and aE e FE) can be rate limiting. As a second example of the study of an enzyme catalysed reaction by 2D-FTNMR, we show a contour plot of the adenylate kinase reaction in Fig. 7. This reaction is of particular interest because the enzyme is found in many tissues and the reaction is an important modulator of high energy phosphate compounds in cells. The reaction: 2ADP+E
Enzyme
No enzyme
0.29 0.15 3.4 2.5 <0.004 co.04
0.06 0.036
ADP exchange rate is about 50% greater that the ATP-ADP exchange rate. The simplest interpretation of this result requires a minimum of two slow steps in the ATP-ADP exchange process. Analysis of the kinetics of this reaction was carried out numerically and the details will be reported elsewherel3. The 2D technique provides an important new means for examining rates of reaction both in vitro and in vivol4. While saturation transfer and inversion transfer remain the techniques of choice in many instances, we anticipate that 2D-FTNMR will become a common method for such biological rate investigations
z AMP+ATP+E
is more complicated in terms of data analysis than the yeast phosphoglucose isomerase reaction because two molecules of ADP are required by the enzyme. -The results of examining the 2D spectra for a sequence of Zmixvalues demonstrates that the AMP-
T
mix
(sec.)
-------/=)O M
1.25
b
1.00
4
-0.75 A
0.50 /‘-‘__O.25
II beta ADP AMP
/,-i_._
60’
0.20
T---~-p---r
F
Fig. 6. Cross section through the 20 plot for the diagonal peak of fructose-6-phosphate.
40bo
’
3000
”
I
2000
”
”
““I’
I
1000
0
Hz
Fig. 7. Contourplot and one-dimensional spectrum for the adenylate kinase catalysed exchange of ADP to AMP and ATP.
152
trends in analyticalchemistry, vol. 3, no. 6,1984
References
7
G. H. Weiss and J. A. Ferretti,
J. Magn. Reson., 55 (1983)
397.
T. Plesser, B. Worster and B. Hess, Eur. J. Biochem.,
98 (1979) 93. G. G. Hammes (Editor), Investigation of Rates and Mechanisms of Reactions, Part 2, Wiley, New York, 1974. D. G. Rhoads and J. M. Lowenstein, J. Biol. Chem., 243 (1968) 3963.
D. G. Gadian, G. K. Radda, R. E. Richards and P. J. Seeley, Biological Applications of Magnetic Resonance, Academic Press, New York, 1979, pp. 463-535. L. M. Jackman and F. A, Cotton, Dynamic NMR Spectroscopy, Academic Press, New York, 197.5. T. R. Brown and S. Ogawa, Proc. Natl. Acad. Sci. U.S.A., 74 (1977) 3627.
8 J. Jeener, B. H. Meier, P. Bachmann and R. R. Ernst, J. Chem. Phys., 71 (1979) 4546. 9 R. S. Balaban and J. A. Ferretti, Proc. Natl. Acad. Sci. U.S.A., 80 (1983) 241.
10 G. H. Weiss and J. A. Ferretti, unpublished results. 11 J. M. Bailey, P. H. Fishman and P. G. Pentcher, J. Biol. Chem., 243 (1968) 4827. 12 K. J. Schray, S. J. Benkovic, P. A. Benkovic and I. A. Rose, J. Biol. Chem., 248 (1973) 2219.
13 H. L. Kantor, J. A. Ferretti and R. S. Balaban, manuscript in preparation. 14 R. S. Balaban, H. L. Kantor and J. A. Ferretti, J. Biol. Chem., 256 (1983) 12787.
PIXE: possibilities in elemental micro- and trace-analysis Rainer P. H. Garten Dortmund, FRG Nonconsumptive microanalysis the nanogram and picogram induced X-ray emission (PIXE). the stage of practical use in analysis.
of solids is feasible down to trace level using partkleThis method has matured to simultaneous multi-element
A number of beam techniques for micro- and trace-element analysis have been developed to determine low-level elemental concentrations and spatial inhomogeneous distributions in solids. A beam of fast ions can be used to create characteristic Xrays from the target atoms within a small sample volume by collision-induced core-level ionization. These projectiles, generally protons of primary energies in the range l-4 MeV, helpfully cause only minor background in the PIXE-spectra, compared with other sources of inner shell ionization. The basic components of PIXE-equipment are the ion-accelerator (ion source with high voltage column), an energy defining magnetic deflection field, and magnetic or electrostatic lenses along the beam pipe, and the high vacuum target chamber with energy-dispersive X-ray detector followed by signal-processing electronics. Contours of X-ray spectra
For a rough estimate orientation on the performance of different X-ray excitation sources, spectra are compared using photon excitation [X-ray fluorescence analysis (XRFA) with an MO transmission tube] and proton excitation of National Bureau of 01659936/84/$02.00.
Standards standard orchard leaves, as shown in Fig. 11.2.According to conclusions drawn from these spectra, a number of more thorough comparative studies clearly revealed that the overall limits of detection in PIXE-analysis are comparable, or superior by a factor of 10, to those gained with conventional XRFAZ-4. The overall figures of merit are governed primarily by the relationship between the cross sections o, for characteristic X-ray production and the atomic number Zi of the analyte i, and by the predominant background-producing process with different excitation sources; but, in addition, they strongly depend on the type of composition, preparation and sampling mode of the individual sample under investigation. This was sometimes ignored, but it is significant considering problems with high ‘blank’ values can be more important than the problems caused by spectral background continuum. General evaluations of method performance are valid only when referenced to a well-defined analytical problem. The main background components are due to Compton scattering from primary radiation within the sample volume in the case of XRFA (the big hump at the high energy end in Fig. lb), and the bremsstrahlung from secondary electrons which are produced in association with the ionization process by primary projectiles in the sample in PIXE (big hump at the low-energy end in Fig. la). According to the secondary origin of these background-producing electrons, the background intensity is rather low. This is emphasized in a comparisons of X-ray spectra induced by either primary protons or electrons (Fig. 2): the electron microprobe (EMP) spectrum is 0 Elsevier Science
Publishers B .V.
148
vol. 3, no. 6, 1984
The measurement of enzyme catalysed rates of reaction by 2D NMR spectroscopy James A. Ferretti and Robert S. Balaban Bethesda, MD, USA
Two-dimensional NMR spectroscopy is useful for studying exchange rates of complex enzyme catalysed reactions in the steady state. The technique provides various technical and interpretive advantages over conventional methods and will be usefulfor studying in vivo processes.
The study of exchange rates of enzyme catalysed reactions is an integral part of the characterization of the enzyme. Such studies also contribute to the understanding of the mechanism and control of metabolic processes which are dictated by the flux of high energy phosphates through enzyme catalysed reactions. These reactions rates have historically been studied by stopped flow’, temperature jump2 and radioactive tracer analysis 3. Both stopped flow and temperature jump analyses require that the system initially be placed in a nonequilibrium state, and its return to equilibrium is monitored to obtain the flux or the rate constants. It is often desirable to study these reactions under equilibrium conditions, where data interpretation is sometimes simpler-s, and where studies in vivo can be carried out. Radioactive tracer analysis, which allows a reaction to be studied at equilibrium, requires the introduction of a radiolabel, followed by an appropriate product assay. Recent advances in nuclear magnetic resonance (NMR) spectroscopy and, in particular, the development of two-dimensional Fourier transform NMR (2D-FTNMR) suggest a marked increase in its use. Our purpose is to review the current use of 2DFTNMR to study enzyme catalysed reactions and to indicate potential future developments in the field. NMR is useful for studying reacting systems in dyn
Preparation
0
T X
namic equilibrium. Other advantages of NMR lie in its capability of following an individual nuclear species and in being a non-invasive technique. This latter feature makes NMR unique attractive for in vivo investigations. Enzyme catalysed rates of exchange have been studied by several NMR techniques. One of the first studies was reported by Gadian et ~1.4, who applied line shapes to the study of the isomerization of glucose-6-phosphate. This technique is applicable to simple systems when the exchange rate is of the same magnitude as the chemical shift difference between the exchanging species. Saturation and inversion transfer of magnetization5,6 have been the methods of choice for studying moderately slow enzymatic reactions, since one can investigate reactions with complex pathways. However, these methods require the application of a second irradiating radio frequency (rf) field which can present technical difficulties on some commercial spectrometers and which can limit selectivity in a crowded spectrum. Also, they rely on detection of small differences in peak areas, which limits precision’. One of the recently introduced 2D-techniques is designed for rate studies. This technique was first suggested by Jeener et al.8 and has been applied to the study of enzyme catalysed reaction@. The experiment, which can be performed on most modern NMR spectrometers with no instrumental modifications, consists of three 90 degree pulses as outlined in Fig. 1. The first pulse, followed by an evolution period t,, is used to frequency label the magnetization and generate a second time variable for the 2D Fourier transformation. This period will represent the spin system before exchange takes place. The second or mixing period involves a second 90 degree pulse, an interval rtiX, and a third 90 degree pulse. Any exchange which takes place during Z,ix is easily
n
Evolution
w
0
T X
Detection
Mixing
tzl
I3
Field Gradient Fig. 1. Basic three-pulse sequence for the 20 exchange rate experiment. 01659936/84/$02.00.
@ Elsevier Science Publishers B .V.
trends in analyticalchemistry, vol. 3, no. 6, 1~4
149
represented after 2D-Fourier transformation. The third period, t2, also called the detection period, is used to record the free induction decay (FID). The advantage of this experiment is that the FID contains information on both the initial and final states of the spin system (Fig. 2). The final state is represented by the precession frequencies after the third 90 degree pulse. To complete the representation of the initial state of the system, the interval t, is systematically increased. Fourier transformation (IT) with respect to both t, and t2:
produces a 2D-spectrum which is a function of the two frequency variables F, and F2. Any species which failed to exchange during Z,ix will have the same frequencies along F, and F2. Conversely, species which exchanged during r,ix will have substrate frequencies along F, and product frequencies along F2. These peaks will appear symmetrically at the intersections of the perpendiculars along Fl and F2 and are termed cross peaks. An example of such a situation is shown in Fig. 3 for the anomerization of the a and ,8 forms of glucose-6-phosphate. The cross peaks represent the degree of anomerization which has taken place during t,ix (2.0 s) and they are equal in volume. The anomerization of glucose-6-phosphate, which occurs in the absence of enzyme, is a first order exchange process: cr-glucose-6-phosphate
ep-glucose-6-phosphate
A unique advantage of the 2D-method
is that under
Fig. 2. Schematic representation of the free induction decay for the three pulse experiment on a two site exchange system.
EVOLUTION PERIOD
t-TM’x+ MIXING PERIOD
DETECTION PERIOD
Fig. 3. Stacp plot showing the results of the anomerization of glucose-6-phosphate in the absence of enzyme. The x,,,~~value was 2.0s.
slow exchange conditions the cross peaks represent exclusively species which have undergone exchange. Thus, in a complex spin system all exchange pathways may be observed simultaneously with a single instrument setting. Use of 2D-FTNMR can result in a substantial saving in time or a significant improvement in the precision of the estimate of the rate parameters. Determination of the rate constants requires estimation of peak volumes at various t,ix values. If cross sectional slices through the peaks are Lorentzian, then the volumes may be estimated directly. Alternatively, the volumes may be estimated by taking horizontal areas at various heights above the baselinelo. Rate constants can be obtained from the experimental data using various procedures. In the simpler situations, such as the glucose-6-phosphate anomerization, where the rate equations can be solved analytically, least squares analysis can be employed. The data, together with the results of curve fitting, are shown in Fig. 4. The rate constants obtained by 2D-FTNMR were in excellent agreement with those obtained by classical optical rotation techniques”. For more complex reacting systems, analyses require the assumption of a reaction mechanism which can be difficult to test. An alternative method of data analysis which is quite simple and is useful for complex systems, such as enzyme catalysed reactions, uses measurement of the initial rate of product formation. Use of this initial rate approximation is quite common in the analysis of stopped flow data’*. The cross peak volumes are measured for short T,ix values. This alternative is particularly attractive be-
in analytical
trends
150
7
MIX
=
1.00
chemistry,
vol. 3, no. 6, 1984
20 ,
SEC.
ti 15
10
5
J-----H,;, 0
F*
2
6
4
Fig. 5. A 20 contour plot showing the anomerization and isomerization of glucose-&phosphate and fructose-&phosphate. The tmixvalue was 1.0 s.
n
; I
Fig. 4. Plot of the volumes of the cross peaks (0) and diagonal peaks (0) as a function oft,,, for the glucose-6-phosphate anomerization.
cause of its simplicity and because the cross peaks represent not only the product but also the origin (substrate) associated with a given product. This method was applied to the study of various enzyme catalysed reactions; two examples are the yeast phosphoglucose isomerase catalysed anomerization and the isomerization of glucose-6-phosphate and fructose-6-phosphate, and the adenylate kinase exchange where adenosine diphosphate goes reversibly to adenosine triphosphate and adenosine monophosphate. A 2D-contour plot for the yeast phosphoglucose isomerase reaction is shown in Fig. 5. Cross peaks are apparent for both the anomerization and isomerization reactions. Separate peaks are not observed for the a and p anomers of fructose-6-phosphate because the exchange rates are large compared with chemical shift differences between the resonances of the individual anomers. Systematic variation of Z,ix yields spectra shown in Fig. 6, where one dimensional slices were taken along F2 at the resonance frequency of the fructose-6-phosphate in the F, dimension (see dotted line in Fig. 5). It is in-
teresting to note that isomerization of fructose-6phosphate to a-glucose-6-phosphate begins immediately, whereas isomerization to /?-glucose-6-phosphate occurs only after an induction period. To determine rate constants in the initial rate approximation, we write: =
Xi
kij
E
tmix --) O
where Zij is the normalized cross peak volume, Xi is the mole fraction of substrate, kij is the rate constant, and E is the concentration of free enzyme in solution. The normalized exchange rates, kijE, are presented in Table I. An additional advantage of the 2D-method using the initial rate approximation is that rates are obtained without any knowledge of the spin lattice relaxation times, T,. With some simple assumptions about the reaction mechanisms, one may provide simple interpretations of rate data from 2D-FTNMR which are not readily available from other experimental techniques. For example, if we write the mechanism of the yeast phosphoglucose isomerase reaction as a+E$aE
=PE r\b 7/ FE
=P+E
lb
F+E where a, p, and F refer to the anomers of glucose-6phosphate and fructose-6-phosphate, respectively, we can identify the rate limiting steps in the reaction. Product initially grows linearly for both the anomerization and isomerization reactions, thereby indicating a single slow step for each process. Since the
trends in analyticalchemistry, vol. 3, no. 6, 1984
151
TABLE I. Rates of reaction catalysed by phosphoglucose
isomerase Rate (set-1) (25 “C)
Reaction
a-Glucose-6-phosphate + P-Glucose-6-phosphate /SGlucose-6-phosphate + a-Glucose-6-phosphate Fructose-6-phosphate + a-Glucose-6-phosphate a-Glucose-6-phosphate + Fructose-6-phosphate Fructose-6-phosphate + /3-Glucose-6-phosphate /3-Glucose-6-phosphate -+ Fructose-6-phosphate
anomerization and isomerization occur at widely differing rates (see Table I), only the steps which involve interconversion among the enzyme-substrate complex and the enzyme-product complex (i.e. a E Sj3E and aE e FE) can be rate limiting. As a second example of the study of an enzyme catalysed reaction by 2D-FTNMR, we show a contour plot of the adenylate kinase reaction in Fig. 7. This reaction is of particular interest because the enzyme is found in many tissues and the reaction is an important modulator of high energy phosphate compounds in cells. The reaction: 2ADP+E
Enzyme
No enzyme
0.29 0.15 3.4 2.5 <0.004 co.04
0.06 0.036
ADP exchange rate is about 50% greater that the ATP-ADP exchange rate. The simplest interpretation of this result requires a minimum of two slow steps in the ATP-ADP exchange process. Analysis of the kinetics of this reaction was carried out numerically and the details will be reported elsewherel3. The 2D technique provides an important new means for examining rates of reaction both in vitro and in vivol4. While saturation transfer and inversion transfer remain the techniques of choice in many instances, we anticipate that 2D-FTNMR will become a common method for such biological rate investigations
z AMP+ATP+E
is more complicated in terms of data analysis than the yeast phosphoglucose isomerase reaction because two molecules of ADP are required by the enzyme. -The results of examining the 2D spectra for a sequence of Zmixvalues demonstrates that the AMP-
T
mix
(sec.)
-------/=)O M
1.25
b
1.00
4
-0.75 A
0.50 /‘-‘__O.25
II beta ADP AMP
/,-i_._
60’
0.20
T---~-p---r
F
Fig. 6. Cross section through the 20 plot for the diagonal peak of fructose-6-phosphate.
40bo
’
3000
”
I
2000
”
”
““I’
I
1000
0
Hz
Fig. 7. Contourplot and one-dimensional spectrum for the adenylate kinase catalysed exchange of ADP to AMP and ATP.
152
trends in analyticalchemistry, vol. 3, no. 6,1984
References
7
G. H. Weiss and J. A. Ferretti,
J. Magn. Reson., 55 (1983)
397.
T. Plesser, B. Worster and B. Hess, Eur. J. Biochem.,
98 (1979) 93. G. G. Hammes (Editor), Investigation of Rates and Mechanisms of Reactions, Part 2, Wiley, New York, 1974. D. G. Rhoads and J. M. Lowenstein, J. Biol. Chem., 243 (1968) 3963.
D. G. Gadian, G. K. Radda, R. E. Richards and P. J. Seeley, Biological Applications of Magnetic Resonance, Academic Press, New York, 1979, pp. 463-535. L. M. Jackman and F. A, Cotton, Dynamic NMR Spectroscopy, Academic Press, New York, 197.5. T. R. Brown and S. Ogawa, Proc. Natl. Acad. Sci. U.S.A., 74 (1977) 3627.
8 J. Jeener, B. H. Meier, P. Bachmann and R. R. Ernst, J. Chem. Phys., 71 (1979) 4546. 9 R. S. Balaban and J. A. Ferretti, Proc. Natl. Acad. Sci. U.S.A., 80 (1983) 241.
10 G. H. Weiss and J. A. Ferretti, unpublished results. 11 J. M. Bailey, P. H. Fishman and P. G. Pentcher, J. Biol. Chem., 243 (1968) 4827. 12 K. J. Schray, S. J. Benkovic, P. A. Benkovic and I. A. Rose, J. Biol. Chem., 248 (1973) 2219.
13 H. L. Kantor, J. A. Ferretti and R. S. Balaban, manuscript in preparation. 14 R. S. Balaban, H. L. Kantor and J. A. Ferretti, J. Biol. Chem., 256 (1983) 12787.
PIXE: possibilities in elemental micro- and trace-analysis Rainer P. H. Garten Dortmund, FRG Nonconsumptive microanalysis the nanogram and picogram induced X-ray emission (PIXE). the stage of practical use in analysis.
of solids is feasible down to trace level using partkleThis method has matured to simultaneous multi-element
A number of beam techniques for micro- and trace-element analysis have been developed to determine low-level elemental concentrations and spatial inhomogeneous distributions in solids. A beam of fast ions can be used to create characteristic Xrays from the target atoms within a small sample volume by collision-induced core-level ionization. These projectiles, generally protons of primary energies in the range l-4 MeV, helpfully cause only minor background in the PIXE-spectra, compared with other sources of inner shell ionization. The basic components of PIXE-equipment are the ion-accelerator (ion source with high voltage column), an energy defining magnetic deflection field, and magnetic or electrostatic lenses along the beam pipe, and the high vacuum target chamber with energy-dispersive X-ray detector followed by signal-processing electronics. Contours of X-ray spectra
For a rough estimate orientation on the performance of different X-ray excitation sources, spectra are compared using photon excitation [X-ray fluorescence analysis (XRFA) with an MO transmission tube] and proton excitation of National Bureau of 01659936/84/$02.00.
Standards standard orchard leaves, as shown in Fig. 11.2.According to conclusions drawn from these spectra, a number of more thorough comparative studies clearly revealed that the overall limits of detection in PIXE-analysis are comparable, or superior by a factor of 10, to those gained with conventional XRFAZ-4. The overall figures of merit are governed primarily by the relationship between the cross sections o, for characteristic X-ray production and the atomic number Zi of the analyte i, and by the predominant background-producing process with different excitation sources; but, in addition, they strongly depend on the type of composition, preparation and sampling mode of the individual sample under investigation. This was sometimes ignored, but it is significant considering problems with high ‘blank’ values can be more important than the problems caused by spectral background continuum. General evaluations of method performance are valid only when referenced to a well-defined analytical problem. The main background components are due to Compton scattering from primary radiation within the sample volume in the case of XRFA (the big hump at the high energy end in Fig. lb), and the bremsstrahlung from secondary electrons which are produced in association with the ionization process by primary projectiles in the sample in PIXE (big hump at the low-energy end in Fig. la). According to the secondary origin of these background-producing electrons, the background intensity is rather low. This is emphasized in a comparisons of X-ray spectra induced by either primary protons or electrons (Fig. 2): the electron microprobe (EMP) spectrum is 0 Elsevier Science
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