Prof. CC Wang and HGPRTase reaction mechanism

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It was a delight to meet Prof. C.C. Wang, a pioneer in studies on the purine salvage enzymes of parasitc protozoans at the ASBMB Chicago in April 2017.

Prof. C.C. Wang and HGPRTase reaction mechanism It was a delight to meet Prof. C.C. Wang, a pioneer in studies on the purine salvage enzymes of parasitc protozoans at the ASBMB Chicago in April 2017. My brief interaction with him led to email exchanges on the catalysis of hypoxanthine-guanine phosphoribosyltranferases (HGPRT). Our chain of emailing abruptly stopped due to my family commitments and 5 months later reading Prof. Liz Hedstrom’s article in ASBMB Today was the shocker that Prof. Wang had died. Prof. C.C. Wang’s life and achievements are better described in the articles listed at the end of this write-up. One of his trainees Prof. Liz Hedstom was my PhD thesis examiner and did an excellent job vetting my work. I mention this to contrast the complete lack of interest in my work shown by my big name Indian examiner. Below is a brief personal and semi-scientific thought on HGPRT catalysis that I was discussing with him. It was during my graduate studies, more than a decade and a half now, that I worked on the enzyme hypoxanthine-guanine-xanthine phosphoribosyltranferase from the malarial parasite Plasmodium falciparum (Pf). Interest in the reaction mechanism of these enzymes peaked in the late 90’s and tapered off dramatically in the 2000’s. The human enzyme catalyzes the phosphoribosylation of hypoxanthine and guanine robustly and that of xanthine very poorly. My studies with Pf HGXPRTases showed that allopurinol, a hypoxanthine analog was a good substrate for the parasite enzyme. Unlike hypoxanthine, guanine or xanthine, allopurinol has no proton at the 7th position that can be abstracted by the enzyme, implying that proton loss has to happen from the N9 or N8 position. It is the N9 position that takes in the phosphoribosyl group. The enzyme therefore, is most likely binding the N9 deprotonated form of allopurinol with a negative charge at N9 and this N9 anion gets phosphoribosylated. This N9 deprotonation can also explain the hypoxanthine and guanine phosphoribosylations as well. The available literature on the reaction mechanism of HGXPRTases has not considered this seriously. Deprotonation is supposed to happen from the N7 position aided by a conserved aspartic acid (D137 in the human enzyme). The argument I am making here is that this deprotonation from N7 is not mandatory for the reaction to proceed and N9 deprotonated form of the purine base is the likely substrate for the enzyme. This is supported by the fact that the rate of the phosphoribosylation reaction increases with increasing pH. This means that the purine phosphoribosylation by the enzyme can take place by two mechanisms; 1. The enzyme mediated

deprotonation at N7 that is documented in the literature (SN1 dissociative mechanism) and 2. The N9 deprotonated form of the base being a substrate, with the enzyme playing a passive role in bringing phosphoribosylpyrophosphate and the negatively charged base in just the right alignment/orientation for the reaction to proceed. The second mechanism that I propose is SN2 like. Prof. Wang suggested a killer experiment to resolve this unambiguously by using 7deazahypoxanthine. Like allopurinol a proton cannot be abstracted from 7-deazahypoxanthine or even 7-deazaguanine. If these two purine analogs can be phosphoribosylated by any

HG(X)PRTases, then all the complicated explanations given in the nearly 2 decade research on the reaction mechanism of HG(X)PRTases are deeply flawed. This” occam’s razor” principle like experiment will resolve my long standing suspicion that HG(X)PRTases follow a SN2 reaction mechanism and not SN1. If any old HG(X)PRTase hands are reading this, go ahead with this most interesting experiment, it is easy to do and falsifiable.,-phd