Enzymes subunits

The regulatory effects of citrate and palmitoyl-CoA are dependent on the phosphorylation state of acetyl-CoA carboxylase. The animal enzyme is phosphorylated at 8 to 10 sites on each enzyme subunit (Figure 25.4). Some of these sites are reg-... 

A model of adenosine monophosphate (AMP) bound to the AMP binding site was built by first overlaying AMP on ZMP in the enzyme subunit C4 (Figure 2). The model was then energy minimized using 500 steps of... 

P. D., Loftus, M., Stuehr, D. J., Expression of human inducible nitric oxide synthase in a tetrahydrobiopterin (H4B)-deficient cell line H4B promotes assembly of enzyme subunits into an active dimmer, Proc. Natl. Acad. Sci. USA 92 (1995), p. 11771-11775... 

Nickel availability to the host plants severely limits the expression of the R. leguminosarum hydrogenase genes in the P. sativum symbiosis (Brito et al. 1994) and probably in other symbioses such as M. loti-L. corniculatus (Brito et al. 2000). This limitation occurs at the level of processing of the enzyme subunits (Brito et al. 1994). It is not clear, however, whether Ni limitation is due to the bacterial or the plant component of the symbiosis. Recent results of nickel transport experiments with intact pea symbiosomes indicate that the peribacteroid membrane is not a specific barrier for Ni transport into the bacteroid (Bascones et al. unpublished). 

The activity of allosteric enzymes is adjusted by reversible binding of a specific modulator to a regulatory site. Modulators may be the substrate itself or some other metabolite, and the effect of the modulator may be inhibitory or stimulatory. The kinetic behavior of allosteric enzymes reflects cooperative interactions among enzyme subunits. 

A wide variety of esters is hydrolyzed with the same Vmax (Table 7.8),37 constant product ratios are found (Table 7.9) stopped-flow studies using p-nitrophenyl phosphate find a burst of 1 mol of p-nitrophenolate ion released per enzyme subunit and the enzyme is covalently labeled by diisopropyl fluorophosphate.38... 

In zinc metalloenzymes. zinc is a selective stoichiometric constituent and is essential for catalytic activity. It is frequently present in numerical correspondence with the number of active enzymatic sites, coenzyme binding sites, or enzyme subunits Removal of zinc results in loss of activity. Inhibition by metal complexing agents is a characteristic feature of zinc metalloenzymes. However, no direct relationship holds between the inhibitory effectiveness of these agents and their affinity for ionic zinc. Although zinc is the only constituent of zinc metalloenzymes in vivo, it can be replaced by other metals m vitro, such as cobalt, nickel, iron, manganese, cadmium, mercury, and lead, as m the case of carboxy-peprida.ses. 

Since the binding constants of Mn2 + to the tight and weak metal ion sites of unadenylylated enzyme are 5.0 x 10 7 and 4.5 x 10 5 M, respectively, the tight site can be selectively populated under conditions where [enzyme] > [Mn2 +]. Figure 24 shows EPR spectra obtained with a solution of 0.79 mil/ enzyme subunit concentration and 0.7 mAf Mn2+ concentration (113). This spectrum represents Mn2+ bound only at the tight sites with no free Mn2 + present. It shows that bound Mn2 + is in a relatively isotropic environment (i.e., the zero-field splitting is small). 

Schlesinger et al. 20) concluded, on the basis of in vitro rates of dimerization, that the dimerization of enzyme subunits in vivo would not be as rapid as observed unless the subunits were compartmentalized in the cell. The in vitro rate of dimerization seemed to be based upon reoxidation and dimerization of reduced monomers and showed a maximum at 65 /ig/ml with respect to protein concentration. The in vivo process may be rather different, however, and later studies by Schlesinger and Barrett 21) with unreduced monomers would seem to change this conclusion because their rates did not have a maximum with respect to protein concentration. 

The overall relative orientation of the secondary structures of an enzyme determines its three-dimensional shape, or tertiary structure. Some enzymes require multiple copies of the same enzyme to function. The individual enzymes cluster into groups of two or more (called dimers, trimers, etc.) and are held together by intermolecular forces. The relative positioning of the separate enzymes in the cluster determines the overall structure, or quaternary structure, of the supramolecular complex. While all enzymes have tertiary structure, only clusters of multiple enzyme subunits have quaternary structure. The overall folded conformation of a protein in its active, catalytic form is called the active or native conformation. 

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. 

This tetrameric enzyme (subunit 36000) has been the subject of several crystallographic studies (summarized in [55]), and information on the dogfish isozyme has been obtained at high resolution [78]. The subunit structure [79] is illustrated in Fig. 18. On the basis of crystallographic findings, a numbering system for the amino acid chain was introduced [80], and has been widely used [55,79,81]. What were already known as Ser-163, Arg-171 and His-195 would simply become Ser-161, Arg-169 and His-193 in the consecutively numbered complete sequence [82]. However, in other parts of the chain (notably around residue 33, residue 187, and the regions 135-147 and 236-252) there were more extensive differences. For simplicity,... 

This dimeric enzyme (subunit 35000) catalyses a reaction similar to the lactate dehydrogenase reaction, and the subunit structures of the enzymes are strikingly similar [83-85] (Fig. 20). Crystallographic [85] and other [86] evidence suggests that the reaction mechanisms are similar. The 4-pro-R hydrogen of NADH is transferred to the Re side of the oxaloacetate to give L-malate [87],... 

Ca + is required for phosphorylase activation in fat bodies of both P, americana (25) and discoidalis (personal observation). Addition of Ca + elevates fat body phosphorylase kinase activity in P. americana (33). and calmodulin inhibitors suppress CC-stimulated trehalose production by the fat body in vitro. However, direct addition of calmodulin to fat body phosphorylase kinase also suppresses the kinase activity. It is proposed that Ca + interacts directly with a calmodulin-like subunit of phosphorylase kinase to activate the enzyme, and the presence of exogenous calmodulin competes with the enzymic subunit for available Ca + (33). These results suggest that the HGHs may influence adipocyte Ca + levels related to phosphorylase activation to promote glycogenolysis for trehalose synthesis. Possibly, HGH-mediated fat body Ca levels may interact with polyphosphoinositides, diacyl glycerol and protein kinase C as second messengers for endocrine message transduction and phosphorylase activation. 

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