Kinetic Discrimination between Two Types of Enzyme Mechanism. Application to Lactose Synthetase

Abstract

Difficulty may arise in distinguishing between ping-pong and sequential enzyme mechanisms, when using double-reciprocal plots of kinetic data obtained at several fixed values of the concentration of the second substrate; this is because the parallel lines diagnostic of the first case may actually be slowly converging as required by the second. An alternative approach is to choose substrate concentrations so that the second substrate concentration occurs in a number of fixed ratios to the first. A ping-pong mechanism predicts a set of straight lines and a sequential mechanism a set of curved lines (parabolas) in double-reciprocal plots. The method is illustrated by its application to bovinemilk lactose synthetase which requires UDP-galactose and N-acetyl-glucosamine as substrates in a sequential mechanism.In enzyme systems where there are two or more substrates, steady-state kinetic data plotted as the reciprocal of the initial velocity against the reciprocal of the concentration of one substrate (a doublereciprocal plot), repeated at several fixed concentrations of a second substrate, will in many cases show one of two basic patterns, either a set of parallel lines or a set of intersecting straight lines. The parallel set is indicative of a ping-pong mechanism and the intersecting set of a sequential mechanism [l].However, occasions arise where it is experimentally difficult to distinguish in double-reciprocal plots between a set of parallel lines and a set of lines which, though intersecting, is converging very slowly in the region of experimental accessibility. Examples of measurements on enzymes with sequential mechanisms, where the data appear consistent with a pingpong mechanism, include pig kidney D-amino acid oxidase [2] and bovine brain hexokinase [3,4].Lactose synthetase from human milk has also shown this phenomenon when the variation of rate with the concentrations of two of the substrates, UDP-galactose and N-acetyl-glucosamine was examined [ 5 ] . In this case the part of the mechanism involving these two substrates was shown to be sequential by using the stratagem of Koster and Veeger [2] which consists of repeating the experiment in the presence of a fixed concentration of an inhibitor (UDP-glucose) that competes with the earlier-adding of the two substrates (UDP-galactose).The purpose of the present report is to describe an alternative method of distinguishing between ping-pong and sequential mechanisms by carrying out experiments in which the concentrations of the two substrates being investigated are varied in a constant ratio, at several different values of the ratio. The practical problem becomes one of deciding whether the lines of a set are straight or curved (parabolic).The method is illustrated by its application to the galactosyl transferase of bovine milk. This enzyme is one of the two protein components of lactose synthetase [6] and catalyses the synthesis of N-acetyllactosamine (LacNAc) :The second component is the milk protein, a-lactalbumin [7], which modifies the monosaccharide-bin...

Bovine Galactosyl Transferase. Substrate . Manganese Complexes and the Role of Manganese Ions in the Mechanism

“…In a ping-pong mechanism, the graph of the results in the presence of UDP-glucose would have remained as a set of parallel lines. Confirmation of this aspect of the mechanism by other kinetic means has already been reported [14]. The effect of the pair of substrates, UDP-galactose and Mn2+, on the velocity was in agreement with the previous reports [4,5] but at variance with the result of Morrison and Ebner [18] for the bovine milk enzyme, under difl'erent conditions of pH, buffer and temperature, and later confirmed by Geren et al [22].…”

“…A choice among rate equations was made on the basis of the lowest experimental variance, i.e. the sum of squares of the differences between each experimental velocity and its calculated value, divided by the difference between the number of observations and the number of parameters being estimated [14]. Computer programmes based on the method of Cleland [13] were used in simple cases, but in general the more effective algorithm of Marquardt [15] proved satisfactory.…”

Determination of the mechanism and kinetic constants for hog kidney γ-glutamyltransferase

“…Extending the 'ratio' method of Tsopanakis & Herries (1975) to this branched non-sequential mechanism, we define the ratio of acceptor/donor concentrations as R = [A]/[D]. We make this substitution after dividing the numerator and denominator of eqn.…”

“…As Tsopanakis & Herries (1975) point out, linear constant-ratio double-reciprocal plots may be misleading if the denominator constant term of the sequential rate equation is small, leading to little curvature in the plots. This is unlikely in this case since Vol.…”

Some properties of 3-hydroxy-3-methylglutaryl-coenzyme A synthase from ox liver

“…l(b) appear parallel again suggests Ping Pong kinetic behaviour. As discussed by Middleton (1974) and Tsopanakis & Herries (1975) concentrations on 3-hydroxy-3-methylglutaryl-CoA synthase activity: (a) at pH7.8, (b) at pH8.7 The assay method is described in the text. It seems that at pH7.8 the proposed acetyl-enzyme does exist to a significant extent, but the curves in Fig.…”