Spinach leaf enzyme

Because of the importance of ADPGlc synthetase for plant starch synthesis, efforts have been made to determine the structure of the enzyme and to relate catalytic and allosteric function to structure. The spinach leaf enzyme is composed of two subunits of 51 and 54 kd mass (5,6). The molecular mass of the native enzyme is 206,000 and presumably is a tetramer composed of two of each subunit. The subunits are antigenically dissimilar, exhibit different peptide patterns on HPLC after trypsin digestion and their N-terminal amino acid sequences are different (7). Thus it is likely that the peptide subunits are products of different genes. 

Pyridoxal-5-phosphate (PLP) has been shown to be an activator analogue of 3-PGA (7,8). It activates ADPGlc synthetic rates about 5- to 6-fold and can overcome P inhibition as does the activator 3-PGA (7,8). Reductive phosphopyridoxylation of the spinach leaf enzyme with labeled PLP gives incorporation into both subunits in equimolar amounts (7,9). The PLP modified enzyme is less dependent on the presence of activator (3-PGA) and is more resistant to inhibition by the allosteric inhibitor, P (7-9). The activator,... 

Figure 4 shows the deduced amino acid sequence from the nucleotide sequence of the two clones and compares it with the complete rice seed enzyme deduced amino acid sequence (9). There is a large amount of identity between the amino acid sequences, corresponding to about 76 . Most notable is the sequence between residues 424-434 in spinach leaf where it has been shown that Lys 431 is the site of chemical modification by PLP (7,9). There is complete agreement of this sequence in the same area with the rice seed enzyme sequence 462-472. Moreover, there is complete identity of the deduced amino acid sequences of amino acids 408-434 in the spinach leaf enzyme 51 kd subunit with amino acids 446-472 of the rice endosperm enzyme subunit. 

Other plant ADP-Glc PPases have been studied in detail and they also have been shown to be composed of two dissimilar subunits. ADP-Glc PPase from potato tuber is composed of two different subunits, 50 and 51 kDa, with oljPj heterotetrameric subunit structure. The small subunit of many higher plant ADP-Glc PPases is highly conserved among plants with 85-95% identity. The maize endosperm ADP-Glc PPase, of molecular mass of 230 kDa, reacts with the antibody prepared against the native spinach leaf enzyme in immunoblot experiments. The enzyme is composed of subunits of 55 and 60 kDa, which would correspond, respectively, to the spinach leaf 51 and 54kDa. " ... 

Phenylglyoxal inactivation of the enzyme can be prevented by 3-PGA or by P , evidence that one or more arginine residues are present in the allosteric sites of the spinach leaf enzyme, and both subunits were labeled... 

The sequences are listed in one letter code and were taken from Ballicora et a/. and Smith-White and Preiss." The Lys residues in bold are those covalehtly modified by pyridoxal-P in theAnabaena and spinach leaf enzymes and are highly conserved in other ADP-GIc PPases. In the case of the potato tuber enzyme the Lys residue was identified via site-directed mutagenesis experiments. The numbers 382 and 419 correspond to the Lys residues in the Anabaena ADP-GIc PPase subunit. The activator sites 1 and 2 are present in the small and large subunits of the plant ADP-GIc PPase. 

An extract from the soluble stromal proteins of purified and intact spinach-leaf chloroplasts was prepared by lysis of the cells in buffer, centrifugation of the suspension of broken cells, and concentration of the supernatant with removal of insoluble material. This extract contained all of the enzymes involved in the condensation of the cyclic moieties of thiamine, thiazole, and pyramine. Thus, the synthesis of thiamine in this extract following the addition of pyramine and putative precursors was a proof that the system had the possibility of building the thiazole. It was found that L-tyrosine was the donor of the C-2 carbon atom of thiazole, as in E. coli. Also, as in E. coli cells, addition of 1 -deoxy-D-f/irco-pen-tulose permitted synthesis of the thiamine structure. The relevant enzymes were localized by gel filtration in a fraction covering the 50- to 350-kDa molecular-mass range. This fraction was able to catalyze the formation of the thiazole moiety of thiamine from 0.1 -mM 1-deoxy-D-t/ireo-pentulose at the rate of 220 pmol per mg of protein per hour, in the presence of ATP and Mg2+. 

Phosphorylase activity in spinach leaf is also distributed in the chloroplastic and cytoplasmic fractions, but in comparable amounts.46,47 The cytoplasmic enzyme has much higher affinity for... 

Figure M. Comparison of the primary sequence of the spinach leaf ADPGlc synthetase with the rice endosperm enzyme deduced amino acid sequence. The non-identities of amino acids between the two enzymes are enclosed in boxes. The start and beginning of the SL1 and SL5 clones are indicated with respect to open reading frame as well as the Hind III restriction nuclease region where the nucleotide sequence has not been determined.

The potato tuber, spinach leaf, and maize endosperm enzymes have small subunit masses of 50,000,51,000, and 54,000, respectively, and large subunit masses of 51,000, 54,000, and 60,000, respectively. The small and large subunits have about 50 to 60% identity with each other and have about 30 to 40% identity with the procaryotic ADPGlc PPases. 

At the DNA level, the isolated genes are dissimilar. For example, in wheat leaf and wheat endosperm, there is only 55.7% identity (Olive et al., 1989) and, on the basis of Southern blot hybridization analyses and restriction enzyme mapping, it is concluded that there are at least two distinct gene families in wheat. For spinach leaf and rice endosperm, there is only approximately a 50% identity (B. S. White and J. Preiss, unpublished results, 1998). 

Holmes and Preiss, 1979) with the enzyme. A similar situation is that of the chloroplastic phosphorylase of spinach leaf (Sivak, 1992). 

Few studies on the localization of the starch biosynthetic enzymes were done before 1978, when it was found that ADPGlc PPase was located exclusively in the chloroplast fraction in both spinach (Mares et al., 1978) and pea (Levi and Preiss, 1978). The first detailed study was done by Okita et al. (1979), in which spinach leaf chloroplasts were isolated either by differential centrifugation (Walker, 1971 see also later) or from protoplasts (Nishimura et al, 1976). These plastid preparations contained essentially all of the activity of the starch biosynthetic enzymes, ADPGlc PPase, starch synthase, and branching enzyme. Subsequently, in guard cells of Commelina communis, Robinson and Preiss (1987) showed that the starch biosynthetic enzymes were present exclusively in the chloroplast fraction. 

ADP-Glc PPase from potato tuber has an intermolecular disulfide bridge that links the two small subunits by the Cys residue it can be activated by reduction of the Cys disul-hde linkage (46). At low concentrations (lOjxM) of 3-PGA, both spinach leaf reduced thioredoxin/ and m reduce and activate the enzyme. Fifty percent activation was observed for 4.5-and 8.7- xM reduced thioredoxin/ and m (47). The activation was reversed by oxidized thioredoxin. Cys is conserved in the ADP-Glc PPases from plant leaves and other tissues except for the monocot endosperm enzymes. In photosynthetic tissues, this reduction may also be physiologically pertinent in the hne regulation of the ADP-Glc PPase. 

Pyridoxal-5-phosphate (PLP) could be considered to have some structural analogy to 3-PGA, and it was found to activate both the enzymes from spinach leaf and Anabaena. In spinach ADP-Glc PPase, PLP bound at Lys440, which is very close to the C-terminal of the small subunit, and bound to three Lys residues in the large subunit. Binding to these sites was prevented by the allosteric effector 3-PGA, which indicated that they are close to or are involved directly in the binding of this activator (100, 101). 

The molecular sizes of the pure enzymes from E. coli Rhodospirillum rubrum and spinach leaf have been... 

Table 2 shows the kinetic constant of both substrates ATP and Glc-l-P, and the allosteric effectors, 3-PGA and Pi for the spinach leaf and potato tuber ADP-Glc PPases. For both enzymes there is a great increase of... 

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