Proteins on substrates

Since the sampling depth of TOF-SIMS in the static mode is approximately 2 nm, a thickness much less than for most proteins, TOF-SIMS can detect information on the conformation and orientation of proteins on substrates. There are two interesting approaches to the conformation and orientation of proteins on substrates which have been proposed in recent publications one [82] is based on SFs primary ions and the gentle ionization effect of gold substrate, and others [83,84] are based on spectrum analysis by PCA. 

Covalent immobilization of proteins on nricrostmctured gold surfaces was studied in [226]. On Arese substrates, which were prepared by pCP aird etching. Are immobilization sites of proteins could be spatially controlled using air amino-reactive SAM. The whole process, i.e. production of Are micropattemed substrate including SAM exchairge aird protein immobilization, took a reasonably small amount of time ( 24 h), providing some flexibility in the experimental work. 

Although FeMo-cofactor is clearly knpHcated in substrate reduction cataly2ed by the Mo-nitrogenase, efforts to reduce substrates using the isolated FeMo-cofactor have been mosdy equivocal. Thus the FeMo-cofactor s polypeptide environment must play a critical role in substrate binding and reduction. Also, the different spectroscopic features of protein-bound vs isolated FeMo-cofactor clearly indicate a role for the polypeptide in electronically fine-tuning the substrate-reduction site. Site-directed amino acid substitution studies have been used to probe the possible effects of FeMo-cofactor s polypeptide environment on substrate reduction (163—169). Catalytic and spectroscopic consequences of such substitutions should provide information concerning the specific functions of individual amino acids located within the FeMo-cofactor environment (95,122,149). 

FIGURE 25.20 Triacylglycerols are formed primarily by the action of acyltransferases on mono- and diacylglycerol. Acyltransferase in E. coli is an integral membrane protein (83 kD) and can utilize either fatty acyl-CoAs or acylated acyl carrier proteins as substrates. It shows a particular preference for palmitoyl groups. Eukaryotic acyltransferases nse only fatty acyl-CoA molecnles as substrates. 

In biological pathways, dehydrations rarely occur with isolated alcohols but instead normally take place on substrates in which the -OH is positioned two carbons away from a carbonyl group. In the biosynthesis of fats, for instance, /3-hydroxybutyry) ACP is converted by dehydration to tram-crotonyl ACP, where ACP is an abbreviation for acyl carrier protein. We ll see the reason for this requirement in Section 11.10. 

The high specificity required for the analysis of physiological fluids often necessitates the incorporation of permselective membranes between the sample and the sensor. A typical configuration is presented in Fig. 7, where the membrane system comprises three distinct layers. The outer membrane. A, which encounters the sample solution is indicated by the dashed lines. It most commonly serves to eliminate high molecular weight interferences, such as other enzymes and proteins. The substrate, S, and other small molecules are allowed to enter the enzyme layer, B, which typically consist of a gelatinous material or a porous solid support. The immobilized enzyme catalyzes the conversion of substrate, S, to product, P. The substrate, product or a cofactor may be the species detected electrochemically. In many cases the electrochemical sensor may be prone to interferences and a permselective membrane, C, is required. The response time and sensitivity of the enzyme electrode will depend on the rate of permeation through layers A, B and C the kinetics of enzymatic conversion as well as the charac-... 

The Na/K ATPase has been extensively purified and characterized, and consists of a catalytic a subunit of around 95 kDa and a glycoprotein 0 subunit of approximately 45 kDa (Skou, 1992). The functional transporter exists as a dimer with each monomer consisting of an a and /3 subunit. Hiatt aal. (1984) have su ested that the non-catalytic jS subunit may be involved in the cottect insertion of the a subunit into the lipid bilayer and, therefore, it is conceivable that a modification of the 0 subunit structure may be reflected by changes in the catalytic activity of the a subunit. Therefore, in studies involving the manipulation of tissue glutathione levels, alterations of intracellular redox state may have an effect on substrate binding at an extracellular site on this ion-translocating protein. 

The so-called midpoint potential, Em, of protein-bound [Fe-S] clusters controls both the kinetics and thermodynamics of their reactions. Em may depend on the protein chain s polarity in the vicinity of the metal-sulfur cluster and also upon the bulk solvent accessibility at the site. It is known that nucleotide binding to nitrogenase s Fe-protein, for instance, results in a lowering of the redox potential of its [4Fe-4S] cluster by over 100 mV. This is thought to be essential for electron transfer to MoFe-protein for substrate reduction.11 3... 

Microbiological Quality of Tofu. Tofu is a protein-rich substrate with pH around 6, hence it is quite susceptible to microbial growth. Traditionally, tofu has been made and consumed in the same day. However, in the United States, tofu may be held at the supermarkets for many days on produce counters before consumption where temperatures are usually 10-15°C. Thus, microbial deterioration becomes a serious problem (23-25). 

Figure 9.3 The effects of varying levels of accessory proteins on CYP2C9 kinetics using diclofenac (a) or (S)-warfarin (b) as substrate probe [219].

There are at least two factors that could influence the turnover rate, the site of metabolism (hot spot) and the affinity of a compound toward these enzymes the protein/ligand (substrate or inhibitor) interaction and the chemical reactivity of the compound towards oxidation. Because of the interaction of the protein with the potential ligand, certain atoms of the compound could be exposed to the heme group, and depending on the chemical nature of these moieties the oxidative reaction will take place at different rates, for example celecoxib is metabolized by CYP2C9 at the... 

Fig. 4.1. Fundamentals of the ubiquitin system. Adapted from Ref [5]. Figure 4.1 shows the fundamentals of the ubiquitin system. (1) Ubiquitin is synthesized in linear chains or as the N-terminal fusion with small ribosomal subunits that are cleaved by de-ubiquitylating enzymes to form the active protein. Ubiquitin is then activated in an ATP-dependent manner by El where a thiolester linkage is formed. It is then transthiolated to the active-site cysteine of an E2. E2s interact with E3s and with substrates and mediate either the indirect (in the case of HECT E3s) or direct transfer of ubiquitin to substrate. A number of factors can affect this process. We know that interactions with Hsp70 can facilitate ubiquitylation in specific instances and competition for lysines on substrates with the processes of acetylation and sumoylation may be inhibitory in certain instances. (2) For efficient proteasomal targeting to occur chains of ubiquitin linked internally through K48 must be formed. This appears to involve multiple...

Dougan, D. a., Mogk, a., Zeth, K., Turgay, K., and Bukau, B. AAA+ proteins and substrate recognition, it all depends on their partner in crime. FEBS Lett 2002, 529, 6-10. 

---