Mechanistic studies of leaving group effects on enzymatic catalysis by methylglyoxal synthase

Abstract

This thesis describes the investigations of leaving group effects on enzymatic catalysis by Methylyoxal synthase (MGS). MGS is a glycolytic enzyme involved m bacterial metabolism which catalyzes the irreversible elimination of dihydroxyacetone phosphate (DHAP). In order to probe the effect of various leaving groups to MGS catalysts, a range of' ’mutant’ substrates have been synthesised where the phosphate group of natural substrate DHAP has been replaced by sulfate, thiosulfate and bromo groups yielding the substtates dthydroxyacetone sulfate (DHAS), dihydroxyacetone thiosulfate (DHATS) and bromohydroxyacetone (BHA). To quantify an overall enzymatic rate acceleration detailed kinetic analysis of the solution, non-enzymatic reactions of these substrates have been made. Rates for elimination and enolization via C-1 and C-3 deprotonation respectively have been quantified for each substrate using high resolution (^1)H NMR spectroscopy for a range of pD values. From the second order rate constants for buffer catalyzed deprotonation, k(_B) (M(^-1)s(^-1)) comparisons can be made with k(_car)/K(_M) (M(^-1)s(^-1)) for the enzymatic reactions at the same pD values. The non-enzymatic reactions of DHAS were monitored in the pD range of 7.3-9.2 using phosphate and quinuclidinone buffers. Second order rate constants, k(_B) (M(^-1)s(^-1)) of 5.60 x 10(^-4), 5.28 X 10(^-3), 5.82 x l0(_-3) and 7.40 x l0(^-3) M(^-1)s(^-1) for C-1 deprotonation and 1.41 x l0(^-3), 2.24 x l0(^-3) and 3.30 x 10(^-3) for C-3 deprotonation show that increasing the pD results m an increase in the rate of buffer catalyzed deprotonation (phosphate catalyzed C-3 deprotonation was not observed at this pD). This is indicative of a general base catalyzed deprotonation mechanism. First order rate constants, k(_M) (s(^-1)) for the buffer independent catalyzed deptotonation reactions of 1.39 x 10' (^5), 1.22 X 10(^-4), 1.26 X 10(^4) and 2.04 x l0(^-4) for C-1 deprotonation and 9.66 x l0(^-5), 1.10 x 10(^-4) and 9.30 X 10(^-5) for C-3 deprotonation show that an increase 0.7 pD units leads to only a 67 % and 12 respective tacrease in the mtes of deprotonation at the C-l md C-3 positions. This is reflected in the relatively level pD rate profile of the reaction. The background, non-enzymatic reactions of DHATS were carried out in acetic acid buffers and DCl solutions, In DCl solution an average K(_int) (s(^-1)) value of 1.33 x l0(^-5) s(^-1) was obtained. For the acetate catalyzed C-1 deprotonation reactions it was found that moving from pD 3.75 to 4.38 leads to an overall 4.5 fold increase in the value of k(_int) (s(^-1)) for the buffer independent deprotonation reaction of the C-1 protons of the molecule. Values of 9.03 x l0(^-5), 1.03 x 10(^-4) and 4.07 x 10(^-4) s(^-1) were obtained for the reactions in acetate 5, 10 and 20 % f(_B) respectively. Accordingly, in the same pD range the values for the average of first order rate constants, k(_av) (s(^-1)) increase by a factor of 4.9 fold. This is believed to represent a hydroxide dependent reaction. An average k(_B) (M(^-1)s(^-1)) of 5 x 10(^-2) M(^-1)s(^-1) was obtained for the acetate catalyzed reaction at the C-1 position of the molecule. Reactions of the C-3 protons of this molecule were not quantifiable by (^-1)H NMR and thus were followed by mass spectrometry which suggests that a potential dimerisation reaction occurs following deprotonation at this position. Reactions of the C-3 protons of BHA were monitored in quinuclidinone buffet, with values of k(_B)(M(^-1)s(^-1)) = 3.35 x 10(^-3)M(^-1)s(^-1) and k(_int) (s(^-1)) = 9.35 x l0(^-5) s(^-1) obtained for the buffer catalyzed reaction and buffer-independent reaction respectively. The C-1 deprotonation reactions of BHA were monitored in acetate buffers (70-90 % f(_B)). An average k(_B) (M(^-1)s(^-1)) of 9.17 X 10(^-5) M(^-1)s(^-1) was obtained for the buffer catalyzed reaction in this pD range (5.18-6.09). In the same pD range values of k(_int) (s') - 3.7 x 10(^-6). 4.7 x l0(^-6) and 1.19 x l0(^-5) s (^1)were obtained. The reactions of DHA and DHAS were monitored m the presence of MGS (19.6 and 78.4 µg/mL). While no reaction was observable for DHA in the presence of enzyme. DHAS was found to undergo C-1 and C-3 deprotonation leading to average k(_cat)/K(_M) values of 56.5 M(^-1)s(^-1) and 43.5 M(^-1)s(^-1) for exchange and elimination respectively. The rate for elimination is approximately 90,000 fold slower than that observed for natural substrate DEHAP, k(_cat)/k(_M) = 5.2 x 10(^-6) M(^-1)s(^-1) indicating that the phosphodianior group, of DHAP is for optimal cataysis.