Cryoenzymic Studies on the Transition‐State Analog Complex Creatine Kinase ‐ ADPMg ‐ Nitrate ‐ Creatine

Abstract

Concomitant with the formation of the transition state analog complex creatine kinase . ADPMg . nitrate . creatine [Milner-White, E. J. and Watts, D. C. (1971) Bioclzem. J. 122, 727-7401 there results a large difference spectrum. The shape of the spectrum was characteristic of tryptophan exposure.The kinetics of formation and final amplitude of the spectrum were studied at -15 "C using a stopped-flow apparatus. The results obtained allowed for a plausible reaction pathway for nitrate fixation: the ordered addition of nitrate and creatine to the binary enzyme-ADPMg complex, a slow protein isomerization and the final addition of a molecule each of nitrate and creatine.The kinetic parameters for the formation of the transition state analog complex were compared with those already known for the overall reaction obtained under the same conditions [Barman, T. E., Brun, A. and Travers, F. (1980) Eur. J. Biochem. 110, 397-4031, This comparison revealed a striking analogy between the two processes. This suggests that the conformation of creatine kinase in the analog complex is very similar to that in the transition state complex on the catalytic pathway.In the preceding paper [I] we used the rapid flowquench apparatus under subzero conditions to search for intermediates in the creatine kinase reaction pathway. Under these conditions the enzyme appeared to be partitioned exclusively between enzyme-substrate and enzyme-product complexes : we could not detect any phosphoenzyme or labile phosphate intermediate down to 4ms. We obtained a complex product (creatine phosphate) progress curve which allowed us to estimate the rate constants pertaining to a simple mechanism. These results make it likely that creatine kinase proceeds via a direct in-line transfer between the participating substrates, as has already been suggested by Milner-White and Watts [2].Another approach towards the study of enzyme mechanisms is to use transition state analogs [3,4]. In the case of creatine kinase there is no simple, single analog inhibitor. However, Milner-White and Watts [2] showed that the combination MgADP, nitrate and creatine is a powerful inhibitor of the enzyme. They suggest that the nitrate binds to the enzyme at the site normally occupied by the transAbbreviations. Cr, creatine; CrP, creatine phosphate. Enzymes. Creatine kinase (EC 2.7.3.2); arginine kinase (EC 2.7.3.3). ferable phosphate and they conclude that the complex enzyme . ADPMg . NO3 . creatine may mimic the putative transition state complex enzyme . ADPMg . PO4 .creatine. Now, such a complex would occupy a position between an enzyme-substrate and an enzymeproduct complex on the overall catalytic reaction. Since we already have information on the rate constants involved in this step, it seemed to us important to investigate further the formation of the enzyme . ADPMg . NO3 . Cr (nitrate complex) and to see what analogies there might be between the two processes.In this paper we study the formation of the creatine kinase . ADPMg ' NOs ' Cr complex at subzero temperatures [...

Changes of Creatine Kinase Secondary Structure Induced by the Release of Nucleotides from Caged Compounds

“…An extremely stable complex can be formed with creatine, CK, MgADP and with some type of anion as NO, (MilnerWhite and Watts, 1971 ;Watts, 1973). This complex called the transition-state-analog complex of CK, can be viewed as an intermediate state in the phosphoryl transfer (Travers and Barman, 1980). Therefore any structural information gained about this transition-state analog could provide more insights into the enzymic mechanism of phosphoryl transfer in CK.…”

“…suggested by ultraviolet spectroscopic and kinetic studies that the conformation of the transition-state-analog complex was very similar than the conformation of the transition-state complex on the catalytic pathway (Travers and Barman, 1980). Therefore any structural alteration of the transition-state-analog complex could provide some clues into the catalytic mechanism.…”

“…Recently, the photoaffinity labeling of CK with 2-azidoadenosine triphosphate and 8-azidoadenosine triphosphate allowed the location of the binding domain for the adenine moiety of ATP within the peptide regions 236-241 and 279-291 of CK (Olcott et al, 1994). The structural changes related either to the enzyme, such as Trp residues (Balny et al, 1985;Grossman et al, 1992;Roustan et al, 1968;Travers and Barman, 1980), or to the substrates such as an isotopically labeled adenosine [y-(S)-"O, "0, '*O]triphosphate (Han-sen and Knowles, 1981), or cobaltous complex CoADP- (Gabriel and Davis, 19771, inanganous complex MnADP-(Reed and Cohn, 1972) and azide, nitrate or thiocyanate anions (Reed et al, 1978) were monitored and provided valuable mechanistic information, in particular on the in-line displacement mechanism of the phosphoryl transfer. To gain more information on the effects of substrate binding on the secondary structure of CK, and on the contribution of single amino acid residue to the mechanism of reaction, we have used reaction-induced infrared difference spectroscopy (RIDS ; Jackson and Mantsch, 1995 ;Mantele, 1993;Rotschild, 1992;Siebert, 1995).…”

ADP‐Binding and ATP‐Binding Sites in Native and Proteinase‐K‐Digested Creative Kinase, Probed by Reaction‐Induced Difference Infrared Spectroscopy

“…This transition-analog complex can be considered as a transition state of the catalytic pathway (Travers and Barman, 1980;Balny et al, 1985) and therefore can provide more insight into the molecular mechanism of phosphoryl transfer. The RIDS of the nicked-CK .…”

Nucleotide Binding Sites in Wild‐Type Creatine Kinase and in W227Y Mutant Probed by Photochemical Release of Nucleotides and inFrared Difference Spectroscopy

“…The changes of ultraviolet absorption spectra of CK produced by the addition of ATP or ADP (with or without creatine and nitrate), suggested that a Trp residue is near the active site (Roustan et al, 1968;Travers and Barman, 1980;Balny et al, 1985). Based on the rotatory dispersion spectra of complexes of CK with ATP or ADP (Kagi et al, 1971), it was proposed that the large Cotton effect observed was caused by the stacking of the adenine purine moiety with one aromatic group of the enzyme.…”