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Markham GD , Bock CL , Schalk-Hihi C
Acid-base catalysis in the chemical mechanism of inosine monophosphate dehydrogenase
Biochemistry. 1999 Apr 6;38(14) :4433-4440
PMID: ISI:000079834100024   
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Inosine-5'-monophosphate dehydrogenase (IMPDH) catalyzes the K+-dependent reaction IMP + NAD + H2O --> XMP + NADH + H+ which is the rate-limiting step in guanine nucleotide biosynthesis. The catalytic mechanism of the human type-II IMPDH isozyme has been studied by measurement of the pH dependencies of the normal reaction, of the hydrolysis of 2-chloro-IMP (which yields XMP and Cl- in the absence of NAD), and of inactivation by the affinity label 6-chloro-purine-ribotide (6-Cl-PRT). The pH dependence of the IMPDH reaction shows bell-shaped profiles for k(cat) and the k(cat)/K-m values for both IMP and NAD, illustrating the involvement of both acidic and basic groups in catalysis. Half-maximal k(cat) values occur at pH values of 7.2 and 9.8; similar pK values of 6.9 and 9.4 are seen in the k(cat)/K-m profile for NAD. The k(cat)/K-m profile for IMP, which binds first in the predominantly ordered kinetic mechanism, shows pK values of 8.1 and 7.3 for acidic and basic groups, respectively. None of the kinetic pK values correspond to ionizations of the free substrates and thus reflect ionization of the enzyme or enzyme-substrate complexes. The rate of inactivation by 6-Cl-PRT, which modifies the active site sulfhydryl of cysteine-331, increases with pH; the pK of 7.5 reflects the ionization of the sulfhydryl in the E.6-Cl-PRT complex. The pKs of the acids observed in the IMPDH reaction likely also reflect ionization of the cysteine-331 sulfhydryl which adds to C-2 of IMP prior to NAD reduction. The k(cat) and k(cat)/K-m values for hydrolysis of 2-Cl-IMP show a pK value of 9.9 for a basic group, similar to that seen in the overall reaction, but do not exhibit the ionization of an acidic group. Surprisingly, the rates of 2Cl-IMP hydrolysis and of inactivation by 6-Cl-PRT are not stimulated by K+, in contrast to the >100-fold K+ activation of the IMPDH reaction. Apparently the enigmatic role of K+ lies in the NAD(H)- dependent segment of the IMPDH reaction. To evaluate the importance of hydrogen bonding in substrate binding, several deamino- and deoxy-analogues of IMP were tested as substrates and inhibitors. Only 2'-deoxy-IMP was a substrate; the other compounds tested were competitive inhibitors with K-i values at most 10-fold greater than the K-D for IMP, illustrating the greater importance of hydrogen-bonding interactions in the chemistry of the IMPDH reaction than simply in nucleotide binding.
Times Cited: 5 English Article 188EP BIOCHEMISTRY-USA