Triosephosphate isomerase from baker’s yeast – ribozyme versus protein

It was previously shown that in baker’s yeast Saccharomyces cerevisiae, transketolase can exist not only free, but in complex with RNA. The complex does not possess transketolase activity [N.K. Tikhomirova, G.A. Kochetov, A new method of isolation and a new form of transketolase from baker’s yeast, Biokhimiia 56 (1991) 1123-1130]. We discovered that this RNA is a ribozyme which catalyzes the interconversion of glyceraldehyde 3-phosphate (G3P) and dihydroxyacetone 3-phosphate (DHAP), i.e. acting as triosephosphate isomerase (TPI). It also catalyzes an unusual reaction of ribose 5-phosphate (R5P) decomposition to G3P and erythrose. TPI-ribozyme was found in baker’s yeast not only in complex with transketolase, but also in free form. Transketolase-RNA complex was easily isolated on an immunoaffi nity column with antibodies to transketolase. TPI-ribozyme consists of 87 nucleotides and has a molecular weight of 26.6 kDa. The optimum of pH-activity is 7.5 for DHAP, 6.7 for R5P and 9.0 for G3P. Km and Vmax are accordingly 0.29 mM and 2.6 U/mg for DHAP, 22 mM and 0.65 U/mg for R5P, 0.05 mM and 4.3 U/mg at pH 7.6 and 0.11 mM and 16 U/mg at pH 9.0 for G3P. These kinetic characteristics are the same for free RNA and in the complex with transketolase. Ki for RNA binding to transketolase was 1.0 μM. Accordingly, the TPI-ribozyme performs a dual function – it shows TPI activity and blocks the work of transketolase, thereby switching the metabolic process to glycolysis. The location of the TPI-ribozyme gene is determined. Blocking the activity of transketolase by ribozyme may be of practical importance in medicine, particularly, in cancer therapy. Research Article Triosephosphate isomerase from baker’s yeast – ribozyme versus protein ON Solovjeva* A.N Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119992 Moscow, Russia Received: 30 July, 2020 Accepted: 17 August, 2020 Published: 18 August, 2020 *Corresponding author: ON Solovjeva, A.N Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119992 Moscow, Russia, Tel: +7 495 939 1456; Fax: +7 495 939 3181; E-mail:

About 100 reviews have been written about these ribozymes.
The review [32], contains a table of RNA classes, their sizes and functions. The review [33], provides a detailed description of the chemical properties of ribozymes, the role of metals and the characteristics of individual ribozymes, as well as the methods used in ribozyme studies. Self-cleaving ribozymes are described in review [23], riboswitches in reviews [34,35]. The latest review of RNase was published in 2009 [28].
Since 2003, 5 articles have been published on ribozymes that are not involved in the biosynthesis of RNA or protein.
They are involved in the reactions of alcoholic fermentation -pyruvate decarboxylase ribozyme [36] and alcohol dehydrogenase ribozyme [37,38] and in glycolysis -aldolase ribozyme [39,40]. Interestingly, free thiamine is used in the pyruvate decarboxylase ribozyme, while thiamine diphosphate is used in the pyruvate decarboxylase protein [36]. Alcohol dehydrogenase ribozyme oxidizes alcohol only in the presence of NAD+ and Zn 2+ [37]. Like alcohol dehydrogenase, a protein, it is able to catalyze a reverse reaction in the presence of NADH and Zn 2+ [38]. Aldolases (protein enzymes) utilize either an enamine mechanism or a Zn 2+ cofactor. Aldolase ribozyme is a Zn 2+ -dependent. Ribozyme, unlike the protein enzyme, can also catalyze a transfer of a biotinylated benzaldehyde derivative to the aldol donor substrate [39]. All were obtained by in vitro selection using an RNA library.
In this paper, we fi rst show the existence of another glycolytic ribozyme, Triosephosphate Isomerase (TPI)ribozyme, which was found in baker's yeast, both free and associated with Transketolase (TK), performing, respectively, two functions -the work of the glycolytic enzyme and blocking the enzyme of the pentose phosphate pathway and accordingly the switching of metabolism to glycolysis. The activity of TPIribozyme is the same as a part of the complex and in free form.
It was previously shown that TK can be isolated from yeast in the form of a complex with RNA [41,42]. G3P, which is formed from DHAP upon catalysis by TPI (enzyme and ribozyme), is one of the substrates of transketolase. Nevertheless, it is not clear why the shutdown of TK is carried out precisely by TPIribozyme.
We suggest that in perspective for most enzymes, ribozymes with a similar function will be found. It is possible that other ribozymes will also be associated with proteins and so will be easy to isolate. The presence of a complex with nucleic acids is known for many enzymes, including those not involved directly in the biosynthesis of nucleic acids and proteins [43][44][45]. For glyceraldehyde 3-phosphate dehydrogenase (GAPDH), the interaction with the hepatitis delta virus RNA was shown [46,47].

Measuring protein and RNA concentrations
The concentration of free TK was determined spectrophotometrically using the absorption coeffi cient A 1% 1-cm of 14.5 at 280 nm [48].

Determination of RNA molecular mass
Initially, the molecular mass of RNA was determined by gel chromatography on a Sephadex G-100 column in 6 M urea.

Measuring of transketolase activity
The catalytic activity of TK was measured at room temperature using spectrophotometer Ultrospec 110 pro by the rate of NAD + reduction in a coupled system with GAPDH [53].
The reaction mixture in fi nal volume 1 mL contained 50 mM glycylglycine pH 7.6, 2.5mM CaCl 2 , 1 mM sodium arsenate, 3 mM dithiothreitol, 0.1 mM ThDP, 1.6 mM NAD + , 3 units of GAPDH and 3.5 mg/mL potassium salt of the phosphopentose mixture that was used as substrate. The reaction was initiated by the addition of TK. Phosphopentose mixture was obtained from R5P [54].

Determination of the affi nity of RNA to TK
50 μg/mL of TK were incubated without RNA and with fi xed RNA concentrations of 0.55, 1.0, and 2.65 μM and varying ThDP concentrations of 0.6-3 μM for 40 min without adding cations to the medium. Under these conditions, Ca 2+ is located in only one active site of the dimeric TK molecule. Therefore, ThDP can be bound with only one of the two active sites [55,56]. The results were processed in reverse coordinates. K i was calculated using the formula K m [I] / (K m app -K m ). Also, 0.5 mg/mL TK (3.4 μM) was incubated for 40 min with 6 μM ThDP without the addition of cations (condition of ThDP binding in one of two active sites). Measuring the activity without the addition of cofactors, we were convinced that it is 50% of the maximum, i.e. ThDP was bound only in the fi rst active site. 6 μM RNA was then added and incubated for 20 min and the activity of TK was measured in the presence of 0.1 mM Ca 2+ and 0.1 mM ThDP.

Measuring of RNA and TPI (protein) activity
The catalytic activity of free RNA and in the complex with TK, as well as the activity of the TPI -protein enzyme was measured by the method [57].
When the activity was measured using DHAP or G3P, their freshly prepared solutions were used. The reaction was started by adding TK-RNA or RNA, after waiting for a zero background.
When the activity was measured using R5P, the reaction mixture in the fi nal volume of 1 mL contained 50 mM glycylglycine, 0.3 mM NADH, 3 mM R5P, 3 units of GPDH, 0.5-2.5 g/mL RNA, pH 7.6.

Erythrose assay
After the total conversion of R5P by RNA in the presence of GPDH and NADH, the reaction mixture was treated with Citation: Solovjeva

Isolation of total RNA from yeast extract
Extraction was carried out from dry yeast with 0.5 M ammonia, as in the isolation of TK. Half of the extract was passed through an anti-TK immunoaffi nity column to bind and thus remove the TK and the TK-RNA complex. ACN 3:1 was added to both extracts to remove proteins.

Partial purifi cation of TPI -protein enzyme
After removing the nucleic acids from the yeast extract by treatment with protamine sulfate and removing the TK-RNA by passing through an immunoaffi nity column with antibodies to TK, dry ammonium sulfate was added to 70% saturation.
The precipitate was collected, dissolved in the same volume, and the protein fraction was collected with saturation of 60-70% ammonium sulfate. The absence of GAPDH activity was verifi ed [59].

Isolation of TK-RNA on a column with IRA-400
To obtain the TK-RNA complex without free TK, the TK isolated on the immunoaffi nity column was passed through the column IRA-400. TK-RNA was eluted with 10 mM potassium phosphate buffer at pH 7.6. Free TK remained bound to the ion exchanger even when passing 500 mM buffer through the column (Figure 1). The TK-RNA complex did not possess transketolase activity, which was also shown earlier [41,42]. ThDP in the complex was absent (It was measured enzymatically). RNA can be separated from TK-RNA by ammonium sulfate and therefore is associated non-covalently. Measuring the activity of TK-RNA in the usual for TK transferase reaction in the presence of cofactors showed its complete absence. This means that RNA was associated with both active sites of TK, that is, two RNA molecules are linked to one dimeric TK molecule. Comparing the concentration of TK and nucleotides in the TK-RNA complex, we calculated that one RNA molecule contains 87 nucleotides, i.e. the mass of RNA is about 30 kDa. The molar extinction coeffi cients calculated from the absorption spectra at different wavelengths are shown in Table 1. A 280 for TK bound to RNA is 6.03, while for free TK this value is 1.45 [48].

Determination of the RNA molecular mass
The molecular mass of RNA determined by gel chromatography on Sephadex G-100 under denaturing conditions was 26.6 kDa ( Figure 3A). Then, the molecular mass of RNA was determined using MALDI-TOF MS analysis. The maximum mass value of 13.766±2 kDa was determined ( Figure  3B). So, it can be assumed that the RNA is double-stranded.
These data require further research.    [60]. Therefore, it can be assumed that RNA also binds directly to TK (perhaps also with the residue Asp 477 ) through its R5P.

Reactions catalyzed by RNA. RNA is TPI-ribozyme
RNA associated with TK catalyzes the interconversion reaction of G3P and DHAP, like the protein enzyme TPI, as well as the unusual reaction of converting R5P to DHAP and erythrose. So, it is a ribozyme.
In Figure 5 it is shown the dependence of the rate of these reactions on the concentration of RNA. The reaction rate and kinetic characteristics were the same for free RNA and RNA in a complex with TK. The values of K m and V max for the reactions with these three substrates are shown in Table 2.
In the reaction of RNA with a fi ve-carbon R5P, a threecarbon G3P is formed. Consequently, it should be expected that glycolaldehyde will be the second reaction product. However, the second product of the reaction was erythrose, which may be formed as a result of the condensation of two glycolaldehyde molecules. Assuming that this is the case, two molecules of RNA-converted R5P would yield one molecule of erythrose, as was confi rmed experimentally. Table 3 shows that the amount of erythrose formed corresponds to the calculated data.  The optimum pH in the reactions of RNA with G3P, DHAP and R5P In Figure 6 are shown the results of the measuring the optimum pH activity in the reactions of RNA with substrates.

Comparison of the properties of TPI-ribozyme -free and in complex with TK and TPI-protein in yeast extract
Total RNA was isolated from yeast extract. A portion of the RNA was passed through an immunoaffi nity column to remove the TK-RNA complex. Before and after removal of TK-RNA from the extract, there was RNA activity with G3P, DHAP and R5P. Consequently, TPI-ribozyme is in the extract not only in the form of a complex with TK, but also in a free form.
An ammonium sulfate fraction containing TPI (protein) was also obtained from the yeast extract and the kinetic constants were measured. The ratio of direct and reverse K m reactions is different for RNA and protein: for RNA, the affi nity for G3P is 6 times higher than for DHAP, and for protein, the affi nity for DHAP is 3 times higher than for G3P. At the same time, the affi nity for G3P is 40 times higher in RNA than in protein (Table 2). Only RNA has the ability to catalyze the reaction with R5P. V max of the salt fraction containing TPI was 20 U/mg.
Previously, it was shown that the specifi c activity of TPI from brever's yeast was 41 U/mg in crude homogenate and 10,000 U/mg in the fi nal purifi cation stage [62]. K m for TPI (enzyme) of two types of yeast is almost the same for both substrates ( Table 2).
The optimum pH of the reactions for RNA and TPI (enzyme) is approximately the same -7.5 in the reaction with DHAP

RNA sequence
An RNA sequence was performed. The results are presented in Table 4 and Scheme 2. The largest and with the same number of readings falls on the genes of the ribosomal RNA RDN37-1 and RDN37-2, located in the 12th chromosome and each 5947 nucleotides in length. Several genes that are located within the genes RDN37-1 and RDN37-2 are also sequenced. These are genes RDN25-1 and RDN25-2 of 3396 nucleotides each and RDN18-1 and RDN18-2 of 1600 nucleotides each, as well as several short genes. It can be assumed that TPI-ribozyme is formed by splicing of some exons and is represented by two forms -TPI-ribozyme1 and TPI-ribozyme2, each about 87 nucleotides in length.

Conclusion
Previously was detected inactive TK from baker's yeast [41,42]. TK activity was absent as a result of it's binding to RNA and was restored when the TK-RNA complex dissociated.
So, RNA binding is non-covalent. Due to the great interest in ribozymes, we tried to determine whether RNA associated with TK possesses enzymatic activity. As it turned out, this RNA is a TPI ribozyme and, in addition, unlike protein TPI, slowly cleaves R5P into DHAP and erythrose.
Currently, there are a lot of ribozymes with diverse functions.
There is no doubt that such ribozymes are actually much more. In the case of TK, TPI-ribozyme not only duplicates the work of the protein enzyme, but also blocks the work of TK, thus switching yeast metabolism from the pentose phosphate pathway to glycolysis. The lack of transketolase activity was seasonal. Maximum of complex TK-RNA was in December.
The reason for this switch remains to be found. But this fact indicates that turning off the functioning of TK by forming its complex with RNA is a real mechanism for switching the pentose phosphate pathway and glycolysis in baker's yeast.
We were unable to determine the nucleotide sequence of the RNA ribozyme. According to preliminary data, this RNA is formed from the loci of two genes -RDN37-1 and RDN37-2 which have the same number of nucleotides (Scheme 2 and Table 4). The fact that the mass of RNA determined on the mass spectrometer is 2 times less than that determined with using gel chromatography (13.283 and 26.6 kDa consequently), indicates the possibility that our RNA is double-stranded.
Perhaps these are two ribozymes with the same function.
It is known that to suppress the growth of cancer cells, inhibition of TK is performed [63,64]. Blocking of TK activity could be carried out by forming its complex with RNA.